Antisense oligonucleotides that bind to exon 51 of human dystrophin pre-mRNA

ABSTRACT

The present invention relates to a therapeutic antisense oligonucleotide which binds to exon 51 of the human dystrophin pre-mRNA to induce exon skipping, and conjugates and compositions thereof for the treatment of DMD.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to GB 1711809.2, filed 21 Jul. 2017,the contents of which is hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 15, 2020, is namedSequence Listing as Filed_5872952.txt and is 94,208 bytes in size.

TECHNICAL FIELD

The present invention relates to a therapeutic antisense oligonucleotidewhich binds to exon 51 of the human dystrophin pre-mRNA to induce exonskipping, and conjugates and compositions thereof. The invention furtherrelates to methods and uses of the antisense oligonucleotide for thetreatment of muscular disorders, specifically for Duchenne MuscularDystrophy.

BACKGROUND

Disruption of alternative splicing underlies many diseases, andmodulation of splicing using antisense oligonucleotides can havetherapeutic implications. Splice-switching antisense oligonucleotides(SSOs) are emerging treatments for neuromuscular diseases, with severalSSOs currently undergoing clinical trials for conditions such as spinalmuscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), whereantisense-mediated exon skipping can restore the open reading frame andallow the synthesis of partly or wholly functional proteins instead ofnon-functional ones.

Duchenne muscular dystrophy (DMD) is one of the most prevalent lethalgenetic disorders in boys worldwide, with an incidence of approx. 1 in3,600-9,337 live male births. DMD is caused by the absence of dystrophinprotein due to mutations in the dystrophin (DMD) gene. The gene encodingthe protein contains 79 exons spread out over more than 2 millionnucleotides of DNA. Any exonic mutation that changes the reading frameof the exon, or introduces a stop codon, or is characterized by removalof an entire out of frame exon or exons or duplications of one or moreexons has the potential to disrupt production of functional dystrophin,resulting in DMD. A less severe form of muscular dystrophy, Beckermuscular dystrophy (BMD) has been found to arise where a mutation,typically a deletion of one or more exons, results in a correct readingframe along the entire dystrophin transcript, such that translation ofmRNA into protein is not prematurely terminated. If the joining of theupstream and downstream exons in the processing of a mutated dystrophinpre-mRNA maintains the correct reading frame of the gene, the result isan mRNA coding for a protein with a short internal deletion that retainssome activity resulting in a Becker phenotype. Deletions of an exon orexons which do not alter the reading frame of a dystrophin protein giverise to a BMD phenotype, whereas an exon deletion that causes aframe-shift will give rise to DMD (Monaco, Bertelson et al. 1988). Ingeneral, dystrophin mutations including point mutations and exondeletions that change the reading frame and thus interrupt properprotein translation result in DMD.

Currently one of the most promising therapeutic avenues is exon skippingusing antisense oligonucleotides (AOs). Exon skipping can restore thereading frame by removing the mutant exon and/or its flanking exon(s)from the DMD pre-mRNA, enabling the production of truncated butpartly-functional dystrophin protein. A majority of DMD patients harbourdeletion mutations and 20% of these are amenable to exon 51 skipping.

In September 2016, the US Food and Drug Administration (FDA)conditionally approved the first DMD antisense drug, eteplirsen (Exondys51), which was developed to exclude exon 51 from mutant DMD. Eteplirsenis an AO modified with a phosphorodiamidate morpholino oligomer(morpholino or PMO), an antisense chemistry that has beenwell-established in terms of its safety and effectiveness. However,eteplirsen remains controversial as there is only weak evidencesupporting the effectiveness of the drug, both in terms of restoringdystrophin protein to therapeutically beneficial levels, and improvingclinical outcomes. The FDA has previously rejected another drugcandidate for DMD exon 51 skipping: the2′-O-methyl-phosphorothioate-based AO ‘drisapersen’. Althoughtherapeutics must ensure the highest possible benefit for the lowestamount of risk, no significant improvements in muscle function weredemonstrated upon treatment with drisapersen, and its use led toconcerns over safety.

Therefore, exon skipping therapies currently face a major challenge inthat their observed efficacy in patients has been very low despite thefact that significant therapeutic effects have been demonstrated in manyanimal studies.

Exon skipping efficiency is largely dependent on the AO target sequence:however, there has been little debate or discussion that the sequencestargeted by eteplirsen and drisapersen might not be the optimal choicesfor exon skipping therapy. Several groups have undertaken large-scale AOscreening efforts to determine effective AO sequences computationallyand empirically. However, the exon skipping effectiveness of designedAOs has not been evaluated both quantitatively and statistically.Although restoring dystrophin protein expression is necessary to improvedystrophic muscle function, the ability of AOs to rescue dystrophinprotein expression has not been reported with sufficient methods ofquantification in previous AO screening studies. Other studies havehighly relied on RT-PCR from primary DMD muscle cells. It is remarkablethat the AO sequences of eteplirsen and drisapersen were determined onlywithin this context.

Thus, the effectiveness of exon 51 skipping therapy could be improved byselecting more optimal AO sequences, and by performing more rigorous AOscreening using a more reliable and direct biological measure—such asrescued dystrophin protein in DMD—for validating the best antisenseoligonucleotides to be taken forward in a clinical trial. It is an aimof one or more aspects of the present invention to address one or moresuch problems in the art.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is providedan antisense oligonucleotide capable of binding to exon 51 of humandystrophin pre-mRNA, wherein binding of the antisense oligonucleotidetakes place entirely within the region between 0 and +89 of the pre-mRNAsequence, and wherein the antisense oligonucleotide comprises at least27 bases.

According to a second aspect of the present invention, there is provideda conjugate comprising an antisense oligonucleotide according to thefirst aspect and a carrier, wherein the carrier is conjugated to theantisense oligonucleotide.

According to a third aspect of the present invention, there is provideda cell loaded with a conjugate of the second aspect.

According to a fourth aspect of the present invention, there is provideda pharmaceutical composition comprising an antisense oligonucleotideaccording to the first aspect, and/or a conjugate according to thesecond aspect, and a pharmaceutically acceptable excipient. According toa fifth aspect of the present invention, there is provided a method oftreating a muscular disorder in a subject, comprising administering aneffective amount of an antisense oligonucleotide capable of binding toexon 51 of human dystrophin pre-mRNA to a subject, wherein binding ofthe antisense oligonucleotide takes place entirely within the regionbetween 0 and +89 of the pre-mRNA sequence, and wherein the antisenseoligonucleotide comprises at least 27 bases.

According to a sixth aspect of the present invention, there is providedan antisense oligonucleotide capable of binding to exon 51 of humandystrophin pre-mRNA for use in the treatment of a muscular disorder in asubject, wherein binding of the antisense oligonucleotide takes placeentirely within the region between 0 and +89 of the pre-mRNA sequence,and wherein the antisense oligonucleotide comprises at least 27 bases.

According to a seventh aspect of the present invention, there isprovided a method of increasing human dystrophin protein expression in acell comprising contacting the cell with an effective amount of anantisense oligonucleotide capable of binding to exon 51 of humandystrophin pre-mRNA, wherein binding of the antisense oligonucleotidetakes place entirely within the region between 0 and +89 of the pre-mRNAsequence, and wherein the antisense oligonucleotide comprises at least27 bases.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a series of antisense oligonucleotidesbinding within the early region at 0 to +89 of exon 51 of the dystrophinpre-mRNA sequence and which have a longer than usual length of at least27 bases, each having remarkable efficiency and effectiveness.

In order to produce the antisense oligonucleotides, the inventorsperformed a study which quantitatively evaluated the effectiveness ofmorpholino-based antisense oligonucleotides for exon 51 skipping using asystematic screening method involving in silico, in vitro, and in vivotests.

The inventors carried out a combination screening using a computationalanalysis to predict exon skipping efficiency of designed antisenseoligonucleotide sequences followed by in vitro tests of morpholinoantisense oligonucleotides in immortalized DMD patient-derived musclecell lines. This research revealed that the beginning of the humandystrophin exon 51 sequence is a very promising target region forinducing exon skipping, specifically the region of 0 to +89 of thesequence. This is notably different from the internal region targeted bythe known eteplirsen and drisapersen antisense therapies.

The antisense oligonucleotides identified from this region were thenoptimised for the most effective restoration of dystrophin production inmuscle cells. Various factors were investigated, including the length ofthe antisense oligonucleotides. Surprisingly, the inventors found thatantisense oligonucleotides binding in this early region are moreeffective when they are longer than many of the known antisenseoligonucleotide sequences against exon 51. Specifically, the inventorsidentified an upward trend correlating effectiveness with the length ofthe antisense oligonucleotide from 27 bases and longer. The inventorshave shown that just a few bases difference means the antisenseoligonucleotide has a significantly different efficiency. Asdemonstrated herein, 30-mer antisense oligonucleotides work up to1.5-fold better than a 25-mer of the same sequence (42% vs. 65% skippingefficiency). Without wishing to be bound by theory, this may be becauselonger sequences can be more specific to the target sequence and lesslikely to cause off-target effects.

It is demonstrated herein that the inventors' optimisation of theseidentified antisense oligonucleotide sequences has enabled efficiency inexon 51 skipping and in rescuing dystrophin protein to increase by up tomore than 12-fold and 7-fold respectively compared to the industrystandard ‘eteplirsen’ sequence. Furthermore, statistically significantin vivo exon 51 skipping by the most effective antisense oligonucleotideidentified through these in vitro screenings was confirmed usingtransgenic mice harbouring the human DMD gene, which has never beenshown for the eteplirsen or drisapersen antisense oligonucleotides.Accordingly, the antisense oligonucleotides described herein are shownto provide an effective therapy and treatment for muscular disorders,especially for the treatment of DMD. These antisense oligonucleotidesare not only providing an alternative therapy into a field of medicinein which only one such drug has been approved for market. They alsoprovide an improved option for treatment which is several times moreeffective at increasing dystrophin protein expression. This is expectedto provide a viable option for treatment for those suffering from DMDand other muscular disorders with strong evidence to support theeffectiveness of the therapy.

For the avoidance of doubt, and in order to clarify the way in which thepresent disclosure is to be interpreted, certain terms used inaccordance with the present invention will now be defined further.

The invention includes any combination of the aspects and featuresdescribed except where such a combination is clearly impermissible orexpressly avoided.

It is noted that where aspects of the invention may refer methods oruses including an antisense oligonucleotide, this may also include aconjugate or pharmaceutical composition comprising an antisenseoligonucleotide as defined herein.

The section headings used herein are for organisational purposes onlyand are not to be construed as limiting the subject matter described.

Antisense Oligonucleotide

The present invention relates to antisense oligonucleotides having alength of at least 27 bases that bind to exon 51 of human dystrophinpre-mRNA within the region of 0 to +89 which can be used to treatmuscular disorders.

Suitably, ‘antisense oligonucleotides’ may be referred to herein as‘AOs’ or ‘oligos’ or ‘oligomers’.

Suitably the antisense oligonucleotide induces skipping of exon 51 ofthe human dystrophin gene.

Suitably the antisense oligonucleotide increases skipping of exon 51 ofthe human dystrophin gene.

Suitably the antisense oligonucleotide allows expression of functionalhuman dystrophin protein.

Suitably the antisense oligonucleotide increases expression offunctional human dystrophin protein.

Suitably, the antisense oligonucleotide comprises at least 28 bases,suitably at least 29 bases, suitably at least 30 bases.

Suitably, the antisense oligonucleotide comprises between 27 and 30bases.

In one embodiment, the antisense oligonucleotide comprises 30 bases.

In one embodiment, the antisense oligonucleotide consists of 30 bases.

Suitably, the binding of the antisense oligonucleotide takes placeentirely within the region between 0 and +88, 0 and +87, 0 and +86, 0and +85, 0 and +84, 0 and +83, 0 and +82, 0 and +81, 0 and +80, 0 and+79, 0 and +78 of the pre-mRNA sequence.

In one embodiment, the binding of the antisense oligonucleotide takesplace entirely within the region between 0 and +78 of the pre-mRNAsequence.

Suitably, the antisense oligonucleotide comprises at least 27 bases ofone of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5),SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises at least 28 bases ofone of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5),SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises at least 29 bases ofone of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5),SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises at least 27 contiguousbases of one of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises at least 28 contiguousbases of one of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises at least 29 contiguousbases of one of the following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide shares at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with one of the following sequences: SEQID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4(Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide shares between 90% and 100%identity with one of the following sequences: SEQ ID NO.1 (Ac0), SEQ IDNO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5(Ac48).

Suitably, the antisense oligonucleotide shares at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% homology with one of the following sequences: SEQID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4(Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide shares between 90% and 100%homology with one of the following sequences: SEQ ID NO.1 (Ac0), SEQ IDNO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5(Ac48).

Suitably, the antisense oligonucleotide may comprise a variant antisenseoligonucleotide which differs from one of the following sequences: SEQID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4(Ac30), or SEQ ID NO.5 (Ac48) by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases.

Suitably, the antisense oligonucleotide may comprise a variant antisenseoligonucleotide which differs from one of the following sequences: SEQID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4(Ac30), or SEQ ID NO.5 (Ac48) by up to 3 bases. Suitably, the antisenseoligonucleotide may comprise a variant antisense oligonucleotide whichdiffers from one of the following sequences: SEQ ID NO.1 (Ac0), SEQ IDNO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5(Ac48) by up to 2 bases. Suitably, the antisense oligonucleotide maycomprise a variant antisense oligonucleotide which differs from one ofthe following sequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ IDNO.3 (Ac26), SEQ ID NO.4 (Ac30), or SEQ ID NO.5 (Ac48) by a single base.Suitably, the antisense oligonucleotide comprises one of the followingsequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide consists of one of the followingsequences: SEQ ID NO.1 (Ac0), SEQ ID NO.2 (Ac5), SEQ ID NO.3 (Ac26), SEQID NO.4 (Ac30), or SEQ ID NO.5 (Ac48).

Suitably, the antisense oligonucleotide comprises SEQ ID NO.1 (Ac0) orSEQ ID NO.5 (Ac48).

In one embodiment, the antisense oligonucleotide comprises SEQ ID NO.1(Ac0).

Suitably, the antisense oligonucleotide consists of SEQ ID NO.1 (Ac0) orSEQ ID NO.5 (Ac48).

In one embodiment, the antisense oligonucleotide consists of SEQ ID NO.1(Ac0).

It will be appreciated that the invention may further include aspectsdirected towards each of the individual antisense oligonucleotidesequences listed in Table 3 i.e. an antisense oligonucleotide comprisingor consisting of any of the sequences listed in Table 3. Furthermore, inaccordance with the second aspect of the invention, a conjugatecomprising an antisense oligonucleotide as listed in Table 3 isenvisaged. Furthermore a pharmaceutical composition in accordance withthe fourth aspect of the invention, comprising an antisenseoligonucleotide as listed in Table 3 or a conjugate thereof isenvisaged. Furthermore a medical use in accordance with the fifth aspectof the invention, comprising an antisense oligonucleotide as listed inTable 3 for the treatment of a muscular disorder is envisaged.Furthermore a method of treatment in accordance with the sixth aspectcomprising an antisense oligonucleotide as listed in Table 3 isenvisaged. Furthermore a method of increasing human dystrophin proteinexpression in a cell in accordance with the seventh aspect comprising anantisense oligonucleotide as listed in Table 3 is envisaged.

Suitably, the antisense oligonucleotide is synthetic, and non-natural.

Suitably, the antisense oligonucleotide may be routinely made throughthe well-known technique of solid phase synthesis. Equipment for suchsynthesis is sold by several manufacturers including, for example,Applied Biosystems (Foster City, Calif.). One method for synthesisingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. Any other means for such synthesis known in the art mayadditionally or alternatively be employed.

Suitably, the antisense oligonucleotide is an antisense oligonucleotideanalogue.

Suitably, the term ‘oligonucleotide analogue’ and ‘nucleotide analogue’refer to any modified synthetic analogues of oligonucleotides ornucleotides respectively that are known in the art.

Suitable examples of oligonucleotide analogues include peptide nucleicacids (PNAs), morpholino oligonucleotides, phosphorothioateoligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonateoligonucleotides, acylphosphonate oligonucleotides and phosphoramiditeoligonucleotides.

Suitably, the antisense oligonucleotide comprises morpholino subunits.Suitably therefore, the antisense oligonucleotide is a morpholinoantisense oligonucleotide.

Suitably, the antisense oligonucleotide comprises morpholino subunitslinked together by phosphorus-containing linkages. Suitably therefore,the antisense oligonucleotide is a phosphoramidate or phosphorodiamidatemorpholino antisense oligonucleotide.

The terms ‘morpholino antisense oligonucleotide’ or ‘PMO’(phosphoramidate or phosphorodiamidate morpholino oligonucleotide) referto an antisense oligonucleotide analog composed of morpholino subunitstructures, where (i) the structures are linked together byphosphorus-containing linkages, suitably one to three atoms long,suitably two atoms long, and suitably uncharged or cationic, joining themorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit, and (ii) each morpholino ring bears a purine orpyrimidine base-pairing moiety effective to bind, by base specifichydrogen bonding, to a base in a polynucleotide.

Suitably, the antisense oligonucleotide comprises phosphorus-containingintersubunit linkages joining a morpholino nitrogen of one subunit to a5′ exocyclic carbon of an adjacent subunit.

Suitably, the antisense oligonucleotide comprises phosphorus-containingintersubunit linkages in accordance with the following structure (I):

wherein:

-   -   Y1 is —O—, —S—, —NH—, or —CH2—;    -   Z is O or S;    -   Pj is a purine or pyrimidine base-pairing moiety effective to        bind, by base-specific hydrogen bonding, to a base in a        polynucleotide; and    -   X is fluoro, optionally substituted alkyl, optionally        substituted alkoxy, optionally substituted thioalkoxy, amino,        optionally substituted alkylamino, or optionally substituted        heterocyclyl.

Optionally, variations can be made to the intersubunit linkage as longas the variations do not interfere with binding or activity. Forexample, the oxygen attached to phosphorus may be substituted withsulfur (thiophosphorodiamidate). The 5′ oxygen may be substituted withamino or lower alkyl substituted amino. The pendant nitrogen attached tothe phosphorus may be unsubstituted, monosubstituted, or disubstitutedwith (optionally substituted) lower alkyl.

Suitably, the synthesis, structures, and binding characteristics ofmorpholino oligonucleotides are detailed in U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337,and PCT Appn. No. PCT/US07/11435.

Binding of the Antisense Oligonucleotide

The present invention relates to an antisense oligonucleotide capable ofbinding within the region 0 to +89 of exon 51 of human dystrophinpre-mRNA.

By ‘capable of binding’ it is meant that the antisense oligonucleotidecomprises a sequence with is able to bind to human dystrophin pre-mRNAin the region stated.

Suitably, the antisense oligonucleotide is complementary to a sequenceof human dystrophin pre-mRNA in the region stated.

Suitably, the antisense oligonucleotide comprises a sequence which iscomplementary to a sequence of human dystrophin pre-mRNA in the regionstated.

The antisense oligonucleotide and a sequence within the region 0 to +89of exon 51 of human dystrophin pre-mRNA are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other andthereby cause exon skipping, suitably exon skipping of exon 51. Thus,‘hybridisable’ and ‘complementary’ are terms which are used to indicatea sufficient degree of complementarity or pairing such that stable andspecific binding occurs between the antisense oligonucleotide and asequence within region 0 to +89 of exon 51 of human dystrophin pre-mRNA.Suitably, the antisense oligonucleotide is sufficiently hybridisableand/or complementary to a sequence within region 0 to +89 of exon 51 ofhuman dystrophin pre-mRNA to induce exon skipping, suitably exonskipping of exon 51. Suitably, the antisense oligonucleotide may not be100% complementary to a sequence within region of 0 to +89 of exon 51 ofhuman dystrophin pre-mRNA. However, suitably the antisenseoligonucleotide is sufficiently complementary to avoid non-specificbinding.

Suitably, the antisense oligonucleotide is at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% atleast 99% complementary to a sequence within the region 0 to +89 of exon51 of human dystrophin pre-mRNA.

It is understood that in order for the antisense oligonucleotide to becapable of binding, it does not require that the entire length of theantisense oligonucleotide binds to the human dystrophin pre-mRNA. Itwill be appreciated that a portion of the antisense oligonucleotide maynot bind to the human dystrophin pre-mRNA, for example the 5′ or the 3′ends of the antisense oligonucleotide. However, in accordance with thefirst aspect, the parts of the antisense oligonucleotide which are boundto the human dystrophin pre-mRNA must fall within the region of 0 to +89of exon 51.

Suitably, therefore, the antisense oligonucleotide is hybridisable to asequence within the region of 0 to +89 of exon 51 of human dystrophinpre-mRNA. Suitably, the antisense oligonucleotide is sufficientlyhybridisable to a sequence within the region of 0 to +89 of exon 51 ofhuman dystrophin pre-mRNA to cause exon skipping of exon 51.

Human Dystrophin

The present invention relates to a therapeutic antisense oligonucleotidefor use in the treatment of muscular disorders, particularly dystrophindisorders such as DMD.

Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of theprotein complex that connects the cytoskeleton of a muscle fibre to thesurrounding extracellular matrix through the cell membrane.

Dystrophin protein contains multiple functional domains. The DMD gene,encoding the dystrophin protein, is one of the longest known human genescovering 2.3 megabases (0.08% of the human genome) at locus Xp21. Theprimary transcript in muscle measures about 2,100 kilobases and takes 16hours to transcribe; the mature mRNA measures 14.0 kilobases. The79-exon muscle transcript codes for a protein of 3685 amino acidresidues. Dystrophin protein contains an actin binding domain and acentral rod domain. This large central domain is formed by 24spectrin-like triple-helical elements of about 109 amino acids, whichhave homology to alpha-actinin and spectrin. The repeats are typicallyinterrupted by four proline-rich non-repeat segments, also referred toas hinge regions. Each repeat is encoded by two exons, typicallyinterrupted by an intron between amino acids 47 and 48 in the first partof alpha-helix 2. The other intron is found at different positions inthe repeat, usually scattered over helix 3. Dystrophin also contains acysteine-rich domain including a cysteine-rich segment (i.e., 15Cysteines in 280 amino acids).

In normal cases, the amino-terminus of dystrophin binds to F-actin andthe carboxy-terminus binds to the dystrophin-associated protein complex(DAPC) at the sarcolemma. The DAPC includes the dystroglycans,sarcoglycans, integrins and caveolin, and mutations in any of thesecomponents cause autosomally inherited muscular dystrophies. Normalskeletal muscle tissue contains only small amounts of dystrophin (about0.002% of total muscle protein), but its absence (or abnormalexpression) leads to the development of a severe and currently incurablesymptoms most readily characterized by several aberrant intracellularsignaling pathways that ultimately yield pronounced myofiber necrosis aswell as progressive muscle weakness and fatigability. The DAPC isdestabilized when dystrophin is absent, which results in diminishedlevels of the member proteins, and in turn leads to progressive fibredamage and membrane leakage. In various forms of muscular dystrophy,such as Duchenne's muscular dystrophy (DMD) and Becker's musculardystrophy (BMD), muscle cells produce an altered and functionallydefective form of dystrophin, or no dystrophin at all, mainly due tomutations in the gene sequence that lead to incorrect splicing. Thepredominant expression of the defective dystrophin protein, or thecomplete lack of dystrophin or a dystrophin-like protein, leads to rapidprogression of muscle degeneration.

The mRNA encoding dystrophin in muscular dystrophy patients typicallycontains out-of-frame mutations (e.g. deletions, insertions or splicesite mutations), resulting in frameshift or early termination of thetranslation process, so that in most muscle fibres no functionaldystrophin is produced.

Suitably, the antisense oligonucleotide triggers exon skipping torestore the reading frame of the dystrophin mRNA. Suitably, theantisense oligonucleotide triggers exon skipping of exon 51 to restorethe reading frame of the dystrophin mRNA. Suitably, restoration of thereading frame restores production of a partially functional dystrophinprotein.

Suitably, the partially functional dystrophin is a truncated dystrophinprotein.

Suitably, the truncated dystrophin protein is the same dystrophinprotein produced in patients suffering from the less severe musculardisorder; BMD.

Muscular Disorder

The present invention relates to the use of therapeutic antisenseoligonucleotides in the treatment of muscular disorders.

Suitably the muscular disorder is selected from any muscular disorderresulting from a genetic mutation.

Suitably the muscular disorder is selected from any muscular disorderresulting from a genetic mutation in a gene associated with musclefunction.

Suitably the muscular disorder is selected from any muscular disorderresulting from a genetic mutation in the human dystrophin gene.

Suitably, the muscular disorder is selected from any muscular dystrophydisorder. Suitably, the muscular disorder is selected from Duchennemuscular dystrophy, Becker muscular dystrophy, congenital musculardystrophy, Distal muscular dystrophy, Emery-Dreifuss muscular dystrophy,Facioscapulohumeral muscular dystrophy, Limb-girdle muscular dystrophy,Myotonic muscular dystrophy, Oculopharyngeal Muscular dystrophy.Suitably, the muscular disorder is Duchenne Muscular Dystrophy (DMD) orBecker Muscular Dystrophy (BMD).

In one embodiment, the muscular disorder is DMD.

Carrier and Conjugate

The present invention also relates to a conjugate of the antisenseoligonucleotide with a carrier.

Suitably, the carrier may comprise any molecule operable to transportthe antisense oligonucleotide into a target cell, suitably into a musclecell.

Suitable carriers may include; peptides, small molecule chemicals,polymers, nanoparticles, lipids, liposomes, exosomes or the like.

Suitably, the carrier is a peptide. The peptide may be selected fromviral proteins such as VP22 (derived from herpes virus tegumentprotein), snake venom protein such as CyLOP-1 (derived from crotamin),cell adhesion glycoproteins such as pVEC (derived from murine vascularendothelial-cadherin protein), Penetratin (Antennapedia homeodomain),Tat (human immunodeficiency virus transactivating regulatory protein) orreverse Tat, for example.

Suitably, the peptide is a cell penetrating peptide.

Suitably, the peptide is an arginine-rich cell penetrating peptide.

The use of arginine-rich peptide carriers is particularly useful.Certain arginine based peptide carriers have been shown to be highlyeffective at delivery of antisense compounds into primary cellsincluding muscle cells (Marshall, Oda et al. 2007; Jearawiriyapaisam,Moulton et al. 2008; Wu, Moulton et al. 2008). Furthermore, compared toother peptides, the arginine peptide carriers when conjugated to anantisense oligonucleotide, demonstrate an enhanced ability to altersplicing of several gene transcripts (Marshall, Oda et al. 2007).Suitably, the arginine-rich cell penetrating peptide may be selectedfrom those carrier peptides described in WO2015075747, WO2013030569,WO2009147368, US20120289457, or US20160237426, for example.

In one embodiment, the arginine rich cell penetrating peptide isselected from those described in WO2013030569 or WO2009147368.

Suitably, the carrier has the capability of inducing cell penetration ofthe antisense oligonucleotide within at least 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% of cells of a given cell culture population. Suitably,the carrier has the capability of inducing cell penetration of theantisense oligonucleotide within at least 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% of muscle cells in a muscle cell culture.

Suitably, conjugation of the carrier to the antisense oligonucleotidemay be at any position suitable for forming a covalent bond between thecarrier and the antisense oligonucleotide or between the linker moietyand the antisense oligonucleotide. For example, conjugation of a carriermay be at the 3′ end of the antisense oligonucleotide. Alternatively,conjugation of a carrier to the antisense oligonucleotide may be at the5′ end of the oligonucleotide. Alternatively, a carrier may beconjugated to the antisense oligonucleotide through any of theintersubunit linkages.

Suitably, the carrier is covalently coupled at its N-terminal orC-terminal residue to the 3′ or 5′ end of the antisense oligonucleotide.

Suitably, the carrier is coupled at its C-terminal residue to the 5′ endof the antisense oligonucleotide.

Optionally, where the antisense oligonucleotide comprisesphosphorus-containing intersubunit linkages, and the carrier is apeptide, the peptide may be conjugated to the antisense oligonucleotidevia a covalent bond to the phosphorous of the terminal linkage group.

Alternatively, when the carrier is a peptide, and the antisenseoligonucleotide is a morpholino, the peptide may be conjugated to thenitrogen atom of the 3′ terminal morpholino group of the oligomer.

Optionally, the carrier may be conjugated to the antisenseoligonucleotide via a linker moiety. Optionally, the linker moiety maycomprise one or more of: an optionally substituted piperazinyl moiety, abeta alanine, glycine, proline, and/or a 6-aminohexanoic acid residue inany combination.

Alternatively, the carrier may be conjugated directly to the antisenseoligonucleotide without a linker moiety.

Suitably the conjugate may further comprise a homing moiety.

Suitably, the homing moiety is selective for a selected mammaliantissue, i.e., the same tissue being targeted by the antisenseoligonucleotide. Suitably, the homing moiety is selective for muscletissue.

Suitably, the homing moiety is a homing peptide.

Suitable homing peptides are disclosed in ‘Effective DystrophinRestoration by a Novel Muscle-Homing Peptide-Morpholino Conjugate inDystrophin-Deficient mdx Mice’ Gao et Mol Ther. 2014 Jul; 22(7):1333-1341, for example.

Suitably, the carrier peptide and the homing peptide may be formed as achimeric fusion protein.

Suitably, the conjugate may comprise a chimeric peptide formed from acell penetrating peptide and a muscle-specific homing peptide.

Optionally, the conjugate may be of the form: carrier peptide-homingpeptide-antisense oligonucleotide or of the form: homing peptide-carrierpeptide-antisense oligonucleotide. Suitably, the antisenseoligonucleotide may be conjugated to a carrier that enhances thesolubility of the antisense oligonucleotide. Suitably the solubility inan aqueous medium. Suitably, a carrier that enhances solubility may beconjugated to the antisense oligonucleotide in addition to a carrieroperable to transport the antisense oligonucleotide. Suitably, thecarrier that enhances solubility and the carrier that transports theantisense oligonucleotide may be formed as a chimeric fusion protein.

Suitable carriers that enhance the solubility of an antisenseoligonucleotide are polymers, such as polyethylene glycol, ortriethylene glycol.

Pharmaceutically Acceptable Excipient

The present invention further relates to a pharmaceutical compositioncomprising the antisense oligonucleotide of the invention or a conjugatethereof, further comprising one or more pharmaceutically acceptableexcipients.

Suitably, the pharmaceutical composition is prepared in a manner knownin the art (as described in Remingtons Pharmaceutical Sciences, MackPubl. Co., Easton, Pa. (1985)), with pharmaceutically inert inorganicand/or organic excipients being used. The term ‘pharmaceuticallyacceptable’ refers to molecules and compositions that arephysiologically tolerable and do not typically produce an allergic orsimilarly untoward reaction when administered to a patient.

Suitably, the pharmaceutical composition may be formulated as a pill,tablet, coated tablet, hard gelatin capsule, soft gelatin capsule and/orsuppository, solution and/or syrup, injection solution, microcapsule,implant and/or rod, and the like.

In one embodiment, the pharmaceutical composition may be formulated asan injection solution.

Suitably, pharmaceutically acceptable excipients for preparing pills,tablets, coated tablets and hard gelatin capsules may be selected fromany of: Lactose, corn starch and/or derivatives thereof, talc, stearicacid and/or its salts, etc.

Suitably, pharmaceutically acceptable excipients for preparing softgelatin capsules and/or suppositories may be selected from fats, waxes,semisolid and liquid polyols, natural and/or hardened oils, etc.

Suitably, pharmaceutically acceptable excipients for preparing solutionsand/or syrups may be selected from water, sucrose, invert sugar,glucose, polyols, etc.

Suitably, pharmaceutically acceptable excipients for preparing injectionsolutions may be selected from water, saline, alcohols, glycerol,polyols, vegetable oils, etc.

Suitably, pharmaceutically acceptable excipients for preparingmicrocapsules, implants and/or rods may be selected from mixed polymerssuch as glycolic acid and lactic acid or the like.

In addition, the pharmaceutical composition may comprise a liposomeformulation which are described in N. Weiner, (Drug Develop Ind Pharm 15(1989) 1523), “Liposome Dermatics” (Springer Verlag 1992) and Hayashi(Gene Therapy 3 (1996) 878).

Optionally, the pharmaceutical composition may comprise two or moredifferent antisense oligonucleotides or conjugates thereof. Optionally,the pharmaceutical composition may further comprise one or moreantisense oligonucleotides or conjugates thereof targeting differentexons, suitably different exons of the human dystrophin pre-mRNA.Optionally, the one or more further antisense oligonucleotides orconjugates thereof may target exons adjacent to exon 51, for example,exon 50 or exon 52 of the human dystrophin pre-mRNA. Suitably, the oneor more antisense oligonucleotides or conjugates thereof targetingdifferent exons of the human dystrophin pre-mRNA are operable, togetherwith the antisense oligonucleotide of the invention, to restore thereading frame of dystrophin mRNA.

Optionally, the pharmaceutical composition may further comprise one ormore antisense oligonucleotides or conjugates thereof targetingdifferent genes. For example, the one or more further antisenseoligonucleotides or conjugates thereof may target myostatin. Such dualtargeting is described in ‘Dual exon skipping in myostatin anddystrophin for Duchenne muscular dystrophy’ Kemaladewi et al. BMC MedGenomics. 2011 Apr. 20;4:36.

Optionally, the one or more further antisense oligonucleotides may bejoined together and/or joined to the antisense oligonucleotide of thefirst aspect.

Optionally, the antisense oligonucleotide and/or conjugate may bepresent in the pharmaceutical composition as a physiologically toleratedsalt. Suitably, physiologically tolerated salts retain the desiredbiological activity of the antisense oligonucleotide and/or conjugatethereof and do not impart undesired toxicological effects. For antisenseoligonucleotides, suitable examples of pharmaceutically acceptable saltsinclude (a) salts formed with cations such as sodium, potassium,ammonium, magnesium, calcium, polyamines such as spermine andspermidine, etc.; (b) acid addition salts formed with inorganic acids,for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (c) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

Optionally, the pharmaceutical composition may comprise, in addition toat least one antisense oligonucleotide and/or conjugate, one or moredifferent therapeutically active ingredients. The one or moretherapeutically active ingredients may be selected from, for example:corticosteroids, utrophin-upregulators, TGF-beta inhibitors, andmyostatin inhibitors.

Suitably, in addition to the active ingredients and excipients, apharmaceutical composition may also comprise additives, such as fillers,extenders, disintegrants, binders, lubricants, wetting agents,stabilizing agents, emulsifiers, preservatives, sweeteners, dyes,flavorings or aromatizing agents, thickeners, diluents or bufferingsubstances, and, in addition, solvents and/or solubilizing agents and/oragents for achieving a slow release effect, and also salts for alteringthe osmotic pressure, coating agents and/or antioxidants. Suitableadditives may include Tris-HCl, acetate, phosphate, Tween 80,Polysorbate 80, ascorbic acid, sodium metabisulfite, Thimersol, benzylalcohol, lactose, mannitol, or the like.

Administration

The present invention relates to a therapeutic antisense oligonucleotideand to a pharmaceutical composition comprising the therapeutic antisenseoligonucleotide which are for administration to a subject.

Suitably, the antisense oligonucleotide and/or pharmaceuticalcomposition may be for topical, enteral or parenteral administration.

Suitably, the antisense oligonucleotide and/or pharmaceuticalcomposition may be for administration orally, transdermally,intravenously, intrathecally, intramuscularly, subcutaneously, nasally,transmucosally or the like.

In one embodiment, the antisense oligonucleotide and/or pharmaceuticalcomposition is for intramuscular administration.

In one embodiment, the antisense oligonucleotide and/or pharmaceuticalcomposition is for intramuscular administration by injection.

An ‘effective amount’ or ‘therapeutically effective amount’ refers to anamount of the antisense oligonucleotide, administered to a subject,either as a single dose or as part of a series of doses, which iseffective to produce a desired physiological response or therapeuticeffect in the subject.

Suitably, the desired physiological response includes increasedexpression of a relatively functional or biologically active form of thedystrophin protein, suitably in muscle tissues or cells that contain adefective dystrophin protein or no dystrophin.

Suitably, the desired therapeutic effects include improvements in thesymptoms or pathology of a muscular disorder, reducing the progressionof symptoms or pathology of a muscular disorder, and slowing the onsetof symptoms or pathology of a muscular disorder. Examples of suchsymptoms include fatigue, mental retardation, muscle weakness,difficulty with motor skills (e.g., running, hopping, jumping), frequentfalls, and difficulty walking.

Suitably, the antisense oligonucleotide or conjugate thereof areadministered at a dose in the range from about 0.0001 to about 100 mgper kilogram of body weight per day.

Suitably, the antisense oligonucleotide or conjugate thereof areadministered daily, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 days, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or onceevery 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

Suitably, the dose and frequency of administration may be decided by aphysician, as needed, to maintain the desired expression of a functionaldystrophin protein. Suitably, the antisense oligonucleotide or conjugatethereof may be administered as two, three, four, five, six or moresub-doses separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Subject

The present invention also relates to treatment of a muscular disorderby administering a therapeutically effective amount of the antisenseoligonucleotide or conjugate thereof to a subject in need thereof.

Suitably the subject has a muscular disorder, as defined above.

Suitably, the subject is mammalian. Suitably the subject is human.

Suitably the subject may be male or female. However, suitably thesubject is male.

Suitably, the subject is any age. However, suitably the subject isbetween the ages of 1 month old to 50 years old, suitably between theages of 1 years old and 30 years old, suitably between the ages of 2years old to 27 years old, suitably between the ages of 4 years old to25 years old.

Increased Exon Skipping and Dystrophin Expression

The present invention relates to a therapeutic antisense oligonucleotidefor use in the treatment of muscular disorder by inducing exon skippingin the human dystrophin pre-mRNA to restore functional dystrophinprotein expression.

Suitably, a ‘functional’ dystrophin protein refers to a dystrophinprotein having sufficient biological activity to reduce the progressivedegradation of muscle tissue that is otherwise characteristic ofmuscular dystrophy when compared to the defective form of dystrophinprotein that is present in subjects with a muscular disorder such asDMD.

Suitably, a functional dystrophin protein may have about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% of the in vitro or in vivobiological activity of wild-type dystrophin. Suitably, a functionaldystrophin protein has at least 10% to 20% of the in vitro or in vivobiological activity of wild-type dystrophin.

Suitably, the activity of dystrophin in muscle cultures in vitro can bemeasured according to myotube size, myofibril organization, contractileactivity, and spontaneous clustering of acetylcholine receptors (see,e.g., Brown et al., Journal of Cell Science. 112:209-216, 1999).

Animal models are also valuable resources for studying the pathogenesisof disease, and provide a means to test dystrophin-related activity. Twoof the most widely used animal models for DMD research are the mdx mouseand the golden retriever muscular dystrophy (GRMD) dog, both of whichare dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol84: 165-172, 2003). These and other animal models can be used to measurethe functional activity of various dystrophin proteins.

Suitably, ‘exon skipping’ refers to the process by which an entire exon,or a portion thereof, is removed from a given pre-processed RNA(pre-mRNA), and is thereby excluded from being present in the mature RNAthat is translated into a protein.

Suitably, the portion of the protein that is otherwise encoded by theskipped exon is not present in the expressed form of the protein.

Suitably therefore, exon skipping creates a truncated, though stillfunctional, form of the protein as defined above.

Suitably, the exon being skipped is an exon from the human dystrophingene, which may contain a mutation or other alteration in its sequencethat otherwise causes aberrant splicing.

Suitably, the exon being skipped is exon 51 of the dystrophin gene.

Suitably, the antisense oligonucleotide is operable to induce exonskipping in dystrophin pre-mRNA.

Suitably, the antisense oligonucleotide is operable to induce exonskipping of exon 51 in dystrophin pre-mRNA.

Suitably, the antisense oligonucleotide is operable to increaseexpression of a functional form of a dystrophin protein in muscletissue, and is operable to increase muscle function in muscle tissue.

Suitably, the antisense oligonucleotide is operable to increase musclefunction by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared tomuscle function in subjects with a muscular disorder such as DMD thathave not received the antisense oligonucleotide.

Suitably, the antisense oligonucleotide is operable to increase thepercentage of muscle fibres that express a functional dystrophin proteinin about at least 1%₅ 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibrescompared to subjects with a muscular disorder such as DMD that have notreceived the antisense oligonucleotide.

Suitably, the antisense oligonucleotide is operable to induce expressionof a functional form of a dystrophin protein to a level of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 25, 40, 45, or 50% of theexpression of dystrophin protein in wild type cells and/or subjects.Suitably, the antisense oligonucleotide is operable to induce expressionof a functional form of a dystrophin protein to a level of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% of the expression of dystrophinprotein in wild type cells and/or subjects.

Suitably, the antisense oligonucleotide is operable to induce expressionof a functional form of a dystrophin protein to a level of at least 10,15, or 20% of the expression of dystrophin protein in wild type cellsand/or subjects.

Suitably, the antisense oligonucleotide is operable to induce exon 51skipping in the dystrophin pre-mRNA to a level of at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

Suitably, the antisense oligonucleotide is operable to induce exon 51skipping in the dystrophin pre-mRNA to a level of at least 60%, 70%,80%, 90%, or 100%.

Suitably, the antisense oligonucleotide is operable to induce exon 51skipping in the dystrophin pre-mRNA to a level of between 60% to 80%.

An ‘increased’ or ‘enhanced’ amount may include an increase that is 1.1,1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or moretimes the amount produced when no antisense oligonucleotide compound(the absence of an agent) or a control compound is administered underthe same circumstances.

Suitably, an ‘increased’ or ‘enhanced’ amount is a statisticallysignificant amount.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain embodiments of the present invention will now be described withreference to the following figures and tables in which:

FIG. 1: Shows in vitro screening of antisense oligonucleotides (AOs) andanalog AOs of eteplirsen (aEte) and drisapersen (aDri) at 10 μM, inimmortalized clonal exon 52-deleted DMD skeletal muscle cells (KM571).Differentiated myotubes were harvested at day 5 following transfection(A) Efficiency of exon 51 skipping as measured by one-step RT-PCR.Representative images are shown. M, 100 bp marker; blank, no RNAtemplate. (B) Efficiency in inducing truncated dystrophin protein asmeasured by quantitative Western blotting with the anti-dystrophinC-terminal antibody. Rescued dystrophin protein levels are calculatedusing calibration curves with healthy 8220 cells. Data represent mean±SD from 3-4 independent experiments. ** p<0.01 vs aEte, † p<0.05 and ††p<0.01 vs aDri, §§ p<0.01 vs all of AOs in (A) and vs Ac0 in (B).

FIG. 2: Shows a time-course analysis of dystrophin exon 51 skipping andprotein in an exon 52-deleted DMD-KM571 cell line transfected with Ac0,Ac48, and analog AOs of eteplirsen and drisapersen at 5 μM. Samples werecollected at days 2 and 11 post-transfection (A) RT-PCR analysis of exon51 skipping. M, 100 bp marker; R, replicate number; blank, no RNAtemplates. (B) Quantification of induced dystrophin protein by Westernblotting with the anti-dystrophin C-terminal antibody. Representativereplicates from 3 independent experiments are shown.

FIG. 3: Shows the dose-dependent effects of Ac0, Ac48, and analogs AOsof eteplirsen and drisapersen in immortalized DMD skeletal muscle cellsas measured by one-step RT-PCR and quantitative Western blotting. DMDskeletal muscle cells were transfected with AOs at 1, 3, and 10 pM andharvested at day 5 post-transfection. (A) and (B) show exon 51 skippingefficiency and expression levels of rescued dystrophin protein,respectively, in DMD muscle cells with exon 52 deletion mutation (ID KM571). Efficacy of skipping exon 51 and rescuing dystrophin proteinexpression is shown in (C) and (D), respectively, in DMD muscle cellsharboring exons 48-50 deletion mutation (ID 6594). Data representmean±SD from 3-7 independent experiments in the KM571 cell line and from3-4 independent experiments in the 6594 cell line. *p<0.05, ** p<0.01 vsaEte; † p<0.05 and †† p<0.01 vs Ac48; § p<0.05, §§ p<0.01 vs aDri in thesame concentration, NS, no significance vs Ac0 at the next dose; ns, nosignificance vs Ac0 at 10 μM. (E) Dose-responsiveness to the AOsanalysed by regression model. Statistical validity of regressionequations in skipping and producing dystrophin protein was p<0.008 andp<0.014, respectively. Plots indicate values of exon skipping ordystrophin protein levels predicted in the regression analysis. Theregression slope and 95% confidence interval (CI) are shown inindividual AOs.

FIG. 4: Shows Immunocytochemistry in immortalized DMD patient-derivedskeletal muscle cells with exon 52 (ID KM571) and exons 48-50 deletionmutations (ID 6594). Cells at day 5 post-transfection with 10 μM Ac0,Ac48, and analog eteplirsen (aEte) were stained with anti-dystrophinC-terminal antibody. Grey lines indicate dystrophin-positive myotubes.White dots indicate nuclei counter-stained with DAPI. *indicatesrepresentative false-positive myotubes due to their contraction ordetachment from the culture plate. Representative images are shown from3 independent experiments. Scale bar: 100 μm.

FIG. 5: Shows Length optimization of the Ac0 morpholino antisenseoligonucleotide. Immortalized DMD muscle cells were transfected with Ac0morpholinos composed of 25-, 26-, 27-, 28-, 29-, and 30-mer lengths. Arepresentative image and quantification of exon 51 skipping induced byAc0 morpholinos at 1 μM (A and B) and 3 μM (C and D) in DMD muscle cellswith exon 52 deletion (KM571) are shown as represented by RT-PCR. (E-H)indicate the results in immortalized DMD cells with exons 48-50deletion. The data are shown from 3 independent experiments.

FIG. 6: shows Exon 51 skipping efficiency induced by Ac0, Ac48, analogAOs of eteplirsen (aEte) and drisapersen (aDri) in primary DMD andhealthy skeletal muscle cells. Differentiated myotubes were transfectedwith Ac0, Ac48, and analog eteplirsen and drisapersen at 10 μM, and thenharvested 3 days later. Exon 51 skipping efficiency as represented byone-step RT-PCR was shown in primary DMD cells with the deletionmutation of exons 45-50 (ID 4546) (A and B) or exons 49-50 (ID 4555) (Cand D), and primary healthy muscle cells (E and F). Data representmean±SD from at least triplicate wells in each condition. M, 100 bpmarker. *p<0.05 and ** p<0.01 vs Ac48, †† p<0.01 vs aEte, §§<0.01 vsaDri.

FIG. 7: shows In vivo efficacy of 30-mer Ac0 antisense morpholinooligonucleotide in the hDMD/Dmd-null mouse model. Exon skipping efficacywas analysed by RT-PCR with tibialis anterior muscles 2 weeks after theintramuscular injection of Ac0 morpholino or analog eteplirsen, aEte (50μg in 30 μL saline). (A) Densitometry analysis of exon 51 skipping asrepresented by a microchip-based capillary electrophoresis system. (B)Averaged percentage of exon 51 skipping efficiency (mean±SE). N=7 ineach group. M, marker; NT, non-treated muscle, UM, upper marker dye; LM,lower marker dye.

FIG. 8: shows In vivo efficacy of 30-mer Ac48 antisense morpholinooligonucleotide in the hDMD/Dmd-null mouse model. Exon skipping efficacywas analysed by RT-PCR with tibialis anterior muscles 2 weeks after theintramuscular injection of Ac48 morpholino or analog drisapersen aDri(50 μg in 30 μL saline). Densitometry analysis of exon 51 skipping asrepresented by a microchip-based capillary electrophoresis system. M,marker; NT, non-treated muscle, UM, upper marker dye; LM, lower markerdye.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or 27 process so disclosed, may be combinedin any combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive.

The invention is not restricted to the details of any foregoingembodiments. The invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed. The reader's attention is directed to all papers anddocuments which are filed concurrently with or previous to thisspecification in connection with this application and which are open topublic inspection with this specification, and the contents of all suchpapers and documents are incorporated herein by reference.

EXAMPLES 1. Materials and Methods 1.1 Design and in Silico Screening ofAOs

413 30-mer and 25-mer AOs targeting exon 51 were designed and analysedusing the AO predictive algorithm we recently developed (see Table 3).Table 3 shows in the columns from left to right; the exon number, thedistance from acceptor splice site, the AO sequence (5′ to 3′), thepredicted skipping %, and the ranking within the screen. The left handAOs are 30mers and the right hand AOs are 25-mers. Based on predictedexon skipping efficiencies, 8 AOs spaced at least 4 bases apart wereselected for in vitro screening (Table 2). Target sequence specificitiesof selected AOs, eteplirsen, and drisapersen were analysed using TheUniversity of California, Santa Cruz Genome Browser(http://genome.ucsc.edu/index.html), confirming that the AO sequencestheoretically do not bind any non-target RNA sequences with 100%identity.

1.2 Antisense Morpholinos

All antisense sequences, including analog AOs of eteplirsen anddrisapersen, were synthesized with the morpholino chemistry by GeneTools (Philomath, Oreg.).

1.3 Cells

Immortalized human skeletal muscle cells derived from three healthysubjects (IDs 8220, CHQ, and KM155) and two DMD patients harbouringdeletion mutations of exon 52 (ID KM571) and exons 48-50 (ID 6594) inthe DMD gene, respectively, were generated by transduction with humantelomerase-expressing and cyclin-dependent kinase 4-expressing vectorsin the Institute of Myology human cell immortalization platform, aspreviously described.33 The three immortalized healthy muscle cell lineswere characterized and the clonal line 8220, which showed the highestdystrophin expression was selected as a positive control to preventoverestimation of rescued dystrophin expression in immortalized DMDcells. Primary skeletal muscle cells derived from DMD patients withdeletion mutations of ex45-50 (ID 4546) and ex49-50 (ID 4555) and ahealthy subject were prepared by the BioBank of Skeletal Muscle, NerveTissue, DNA and cell lines.

1.4 AO Transfection

To mimic as closely as possible the in vivo effects of AO-mediated exonskipping therapy, mature, differentiated myotubes expressing sufficientlevels of DMD mRNA were used for in vitro screening. Cells were culturedin proliferation conditions with growth medium (GM): DMEM/F12 withskeletal muscle supplement mix (Promocell, Heidelberg, Germany), 20%fetal bovine serum (Life Technologies, Waltham, MA), and antibiotics (50units penicillin and 50 μg/ml streptomycin, Life Technologies, Waltham,Mass.). Immortalized and primary DMD skeletal muscle cells were seededat 1.7×104/cm2 and 2.2×104/cm2, respectively, in collagen type I-coated12 or 24-well culture plates. Two days after seeding, at approximately80-90% confluence, GM was replaced with differentiation medium (DM):DMEM/F12 with 2% horse serum (GE Healthcare, Chicago, Ill.), 1×ITSsolution (Sigma, St. Louis, Mo.), and antibiotics. After three days inDM, cells were transfected with AO at 1, 3, 5 or 10 μM containing 6 μMof Endo-porter transfection reagent (Gene Tools, Philomath, Oreg.)(concentrated AOs at 1 mM were incubated at 65° C. for 10 min justbefore diluting with DM). Two days following AO transfection,AO-containing DM was replaced with regular DM. Cells were harvested atday 2, 5, or 11 after AO transfection (day 5, 8 or 14 followingdifferentiation).

1.5 Mice

Animal studies were approved by the Animal Care and Use Committee at theUniversity of Alberta, Children's National Medical Center, and NationalCenter of Neurology and Psychiatry (NCNP). Male and female Dmd exon52-deficient mdx5242 and wild-type mice (Jackson Laboratory, Bar Harbor,Me.) with a C57BL/6J background were prepared at age 4-8 weeks. Dmdmutation in affected mice was confirmed by genotyping with PCR. Atransgenic mouse model harboring the human DMD gene and lacking themouse Dmd gene (hDMD/Dmd-null mouse) was generated by cross-breedingmale hDMD mice (Jackson Laboratory, Bar Harbor, Me.) with femaleDmd-null mice.

1.6 Intramuscular Injection

Mouse version morpholinos of AcO, Ac48, eteplirsen or drisapersen at 5or 20 μg in 40 μL saline were intramuscularly injected into tibialisanterior (TA) muscle under inhalation anesthesia with isoflurane aspreviously described.43 Fifty-μg of Ac0 morpholino and analog eteplirsenin 30 μL saline was injected into TA muscles of hDMD/Dmd-null mice. Allmuscle samples were harvested 2 weeks after intramuscular injection.

1.7 Exon Skipping Analysis by RT-PCR

Total RNA was extracted with Trizol (Invitrogen, Waltham, Mass.) aspreviously described. RT-PCR to detect dystrophin mRNA was performedwith the SuperScript III One-Step RT-PCR System (Invitrogen, Waltham,Mass.) and 0.2 μM of forward and reverse primers (see Table 1) for 200ng and 320 ng of total RNA in immortalized and primary skeletal musclecells, respectively. Primers were designed using Primer3Plus softwareand their specificity was confirmed in healthy human skeletal musclecells (line 8220). The RT-PCR conditions were as follows: 50° C. for 5minutes; 94° C. for 2 minutes; 35 cycles at 94° C. for 15 seconds, 60°C. for 30 seconds, and 68° C. for 35 seconds; and 68° C. for 5 minutes.PCR products were separated on a 1.5% agarose gel and visualized by SYBRSafe DNA Gel Stain (Invitrogen, Waltham, Mass.). Using ImageJ software(NIH) or the MCE-202 MultiNA system (Shimadzu, Kyoto, Japan), theefficiency of exon 51 skipping was calculated using the followingformula:

${exon}\mspace{14mu} 51\text{-}{skipped}\mspace{14mu}{transcript}\mspace{14mu}{intensity}\text{/}\left( {{native} + {intermediate} + {{exon}\mspace{14mu} 51\text{-}{skipped}\mspace{14mu}{transcript}\mspace{14mu}{intensities}}} \right) \times 100\mspace{11mu}(\%)\frac{{Ex}\; 51{skippedtranscript}}{{Native} + {{Ex}\; 51{skippedtranscript}}} \times {{d(\%)}.}$

Unknown top bands above the native band, possibly coming from unexpectedsplicing events, were excluded from quantification of skippingefficiency. The sequences of the PCR products were confirmed with BigDye Terminator v3.1 (Applied Biosystems, Waltham, Mass.). GAPDH or 18Sribosomal RNA was used as an internal control.

TABLE 1 Name Sequence (5′→3′) Purpose Ex49/50_94-10_hDMD_FwdCAGCCAGTGAAGAGGAAGTTAG Immortal KM571 DMD cells with ex52 del.SEQ ID NG. 16 Ex53_80-99_hDMD_Rv CCAGCCATTGTGTTGAATCCPrimary DMD and healthy cells SEQ ID NG. 17 hDMDIDmd-null miceEx47_60-79_hDMD_Fwd AGGACCCGTGCTTGTAAGTGImmortal 6594 DMD cells with ex48-50 del. SEQ ID NO. 18Ex52_83-105_hDMD_Rv GATTGTTCTAGCCTCTTGATTGCPrimary 4555 DMD cells with ex49-50 del. SEQ ID NO. 19Ex43/44_167-12_hDMD_Fwd GACAAGGGCGATTTGACAG SEQ ID NO. 20Ex52_83-105_hDMD_Rv GATTGTTCTAGCCTCTTGATTGCPrimary 4546 DMD cells with ex45-50 del. SEQ ID NO. 19Ex49/50_94-10_hDMD_Fwd CAGCCAGTGAAGAGGAAGTTAG Primary healthy cellsSEQ ID NO. 16 Ex52_83-105_hDMD_Rv GATTGTTCTAGCCTCTTGATTGChDMD/Dmd-null mice SEQ ID NO. 19 mDmd_ex49_83-102_FwdCAAGCACTCAGCCAGTGAAG SEQ ID NO. 21 deletion mDmd_ex53_81-100_RvTCCAGCCATTGTGTTGAATC mdx52 mice with ex52 SEQ ID NO. 22hGAPDH_662-81_Fwd TCCCTGAGCTGAACGGGAAG SEQ ID NO. 23control hGAPDH_860-79_Rv GGAGGAGTGGGTGTCGCTGT Internal SEQ ID NO. 24h18S_760-82_Fwd TCGATGCTCTTAGCTGAGTGTCC SEQ ID NO. 25control h18S_1039-58_Rv TGATCGTCTTCGAACCTCCG Internal SEQ ID NO. 26

1.8 Western Blotting

Cells were harvested with RIPA buffer (Thermo Scientific, Waltham,Mass.) containing cOmplete, Mini, EDTA-free protease inhibitor cocktail(Roche, Basel, Switzerland), and then homogenized by passing through a21-gauge needle 10 times. The supernatants as loading samples wereprepared by centrifugation at 14,000 g for 15 min at 4° C. Protein frommuscle tissues were prepared as previously described. Proteinconcentrations were adjusted using the Bradford assay with supernatantsdiluted 100 times with distilled water. Proteins in a sample buffercontaining 10% SDS, 70 mM Tris-HCl, pH 6.8, 5 mM EDTA, 20% glycerol,0.004% bromophenol blue and 5% 2-mercaptoethanol were heated at 70° C.for 10 min. Western blotting was then done as previouslydescribed.32,43,44 Twelve-μg and thirty-μg from cells and tissues,respectively, were used for sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. Blots were incubated with a rabbit polyclonal antibodyagainst dystrophin C-terminal (1:2500, ab15278, Abcam, Cambridge, UnitedKingdom) in the blocking solution or DYS1 antibody against dystrophinrod domain (1:400, Leica Biosystems, Buffalo Grove, Ill.) for 1 hour atroom temperature. The primary antibody was reacted with HRP-conjugatedanti-rabbit or mouse IgG H+L antibody (1:10,000, Bio-Rad, Hercules,Calif.). Expression levels of dystrophin protein induced by AOs werequantified using calibration curves (R2=0.93-0.99) from dystrophinprotein of healthy 8220 skeletal muscle cells diluted with protein fromnon-treated DMD cells, or wild-type mouse using ImageJ (NIH).Alpha-tubulin was detected on the same membrane as a loading control.Myosin heavy chain (MyHC) on post-transferred gels was stained withCoomassie Brilliant Blue (Bio-Rad, Hercules, Calif.) as a loadingcontrol/differentiation marker.

1.9 Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 5 min at room temperature.After washing with PBS containing 0.01% Triton X-100, cells were blockedwith 10% goat serum (Life Technologies, Waltham, Mass.) in PBS with0.05% Triton X-100 for 20 min and then incubated with anti-dystrophinC-terminal (ab15278) or rod-domain (DYS1) antibody at 1:50 dilution inblocking solution overnight at 4° C. Dystrophin signals were detectedwith Alexa 488- or 594-conjugated secondary antibody (1:500). Desmin(1:80, Abcam, Cambridge, United Kingdom) and MyHC-fast type (1:30, LeicaBiosystems, Buffalo Grove, Ill.) were detected to confirm myogenicdifferentiation of cells. Cells were stored in SlowFade Gold AntifadeMountant with DAPI (Invitrogen, Waltham, Mass.) at 4° C. until analysed.

1.10 Immunohistochemistry

Dystrophin-positive muscle fibers on cryosections from TA muscles ofnon-treated and treated mdx52 mice were detected with the ab15278antibody as previously described. Signal intensity of dystrophin in thetreated mice was compared with that in wild-type using neutral densityfilters (Eclipse TE 2000-U, Nikon, Tokyo, Japan).

1.11 Statistical Analysis

For determining the significance of efficiencies in exon skipping anddystrophin protein rescue, data sets were prepared from at least threeindependent experiments in immortalized cells, triplicate wells inprimary cells, and 3-7 mice. The statistical analysis between AO-treatedgroups was performed by one-way ANOVA followed by a post hocTukey-Kramer multiple comparison test. Simple linear regression analysiswas performed for dose-responsiveness to AOs. Statistical significancewas set at p<0.05 for all analyses.

2. Results 2.1 In Silico Screening of AOs for Exon 51 Skipping

We designed total 413 AOs: 204 and 209 AOs with 30-mer and 25-merlengths, respectively, which cover all possible target sites in DMD exon51 (see Table 3). Our exon skipping efficiency algorithm (‘In SilicoScreening Based on Predictive Algorithms as a Design Tool for ExonSkipping Oligonucleotides in Duchenne Muscular Dystrophy’ Echigoya etal. PLOS ONE March 2015) predicted that the highest efficiency for exon51 skipping was 80.5% for 30-mer AOs, and 41.2% for 25-mer AOs in theinitial 5′ site of exon 51. In silico screening indicated a very lowexon skipping efficiency for the 30-base region targeted by eteplirsen(23.7%), which was ranked 92nd in all 413 AO candidates tested. It isnoted that the drisapersen target site is completely encompassed by thatof the 30-mer eteplirsen.

2.2 Characterization of Immortalized Clonal Healthy and DMD SkeletalMuscle Cell Lines

Significant issues in preclinical testing with primary DMD muscle cellsinclude low purity of muscle cells and insufficient amounts of mutantdystrophin mRNA, which present problems when trying to test AO efficacy.To overcome these hurdles, we generated immortalized clonal skeletalmuscle cells from 3 healthy subjects and 2 DMD patients with exon 52(ex52) and ex48-50 deletion (del.) mutations (IDs KM571 and 6594,respectively). All immortalized skeletal muscle cell lines testedexpressed easily detectable dystrophin mRNA from day 3 after inductionof differentiation. To avoid overestimation of dystrophin protein levelsinduced by AOs in DMD cells, we selected a cell line (ID 8220) with thehighest level of dystrophin protein among three immortalized healthyskeletal muscle cell lines as determined by Western blotting to serve asa positive control. Dystrophin protein expression in the 8220 cell linewas also confirmed by immunocytochemistry.

2.3 In Vitro Screening of Exon 51 Skipping AOs

Based on the in silico screening results, we selected eight 30-mer AOs,including both high-ranking and low-ranking sequences, spaced at least 4bases apart from each other for in vitro screening (Table 2). In thepresent study, all tested AOs, including eteplirsen and drisapersensequences, were synthesized using the morpholino chemistry that has beendemonstrated to be well-tolerated in patients enrolled in clinicaltrials. Here, we termed control morpholino oligonucleotides having thesame sequences as eteplirsen and drisapersen (produced by Gene Tools) as“analog eteplirsen” and “analog drisapersen”. In RT-PCR, 5 of ourmorpholino AOs (Ac0, Ac5, Ac26, Ac30 and Ac48) at 10 μM showedsignificantly higher skipping efficiency compared to analog eteplirsenand drisapersen in immortalized DMD skeletal muscle cells harboring ex52del. (FIG. 1A). Of the tested AOs, Ac0 in particular had the highestskipping efficiency, reaching up to 72%, which was 4 and 25 times moreefficient than analogs of eteplirsen and drisapersen, respectively. InWestern blotting, Ac0 also induced the highest levels of dystrophinprotein, reaching up to 16% of levels in the healthy control cell line,followed by Ac48 at 13% (FIG. 1B). Interestingly, the two AOs, Ac0 andAc48, with the highest skipping efficiency when tested were not thosepredicted to be the best from the algorithm.

TABLE 2 Length Distance Predicted Name Oligo sequence (5 to 3′) (mer)from Ac Skip % Ranking hEx51_Ac9 CCACAGGTTGTGTCACCAGAGTAACAGTCT 30 980.5  1 SEQ ID NO. 13 hEx51_Ac0 GTGTCACCAGAGTAACAGTCTGAGTAGGAG 30 0 80.1 2 SEQ ID No.1 hEx51_Ac5 AGGTTGTGTCACCAGAGTAACAGTCTGAGT 30 5 73.0  4SEQ ID NO. 2 hEx51_Ac26 GGCAGTTTCCTTAGTAACCACAGGTTGTGT 30 26 66.3 12SEQ ID NO. 3 hEx51_Ac30 AGATGGCAGTTTCCTTAGTAACCACAGGTT 30 30 55.5 25SEQ ID NO. 4 Eteplirsen CTCCAACATCAAGGAAGATGGCATTTCTAG 30 65 23.7 67SEQ ID NO. 6 hEx51_Ac48 ATGGCATTTCTAGTTTGGAGATGGCAGTTT 30 48 10.6 128 SEQ ID NO. 5 hEx51_Ac141 TTATAACTTGATCAAGCAGAGAAAGCCAGT 30 141  1.8 142 SEQ ID NO. 14 hEx51_Ac207 atacCTTCTGCTTGATGATCATCTCGTTGA 30 207 NA NASEQ ID NO. 15 Drisapersen TCAAGGAAGATGGCATTTCT 20 67 NA NA SEQ ID NO. 7hEx51_Ac0-29mer TGTCACCAGAGTAACAGTCTGAGTAGGAG 29 0 NA NA SEQ ID NO. 8hEx51_Ac0-28mer GTCACCAGAGTAACAGTCTGAGTAGGAG 28 0 NA NA SEQ ID NO. 9hEx51_Ac0-27mer TCACCAGAGTAACAGTCTGAGTAGGAG 27 0 NA NA SEQ ID NO. 10hEx51_Ac0-26mer CACCAGAGTAACAGTCTGAGTAGGAG 26 0 NA NA SEQ ID NO. 11hEx51_Ac0-25mer ACCAGAGTAACAGTCTGAGTAGGAG 25 0 33.3  10^(a)SEQ ID NO. 12 Ac, acceptor splice site. Uncapitalized nucleotidesindicate intronic sequence. ^(a)the ranking in 25-mer AOs.

2.4 Time-Course Analysis With Ac0, Ac48, and Analog AOs of Eteplirsenand Drisapersen

The persistent effects of Ac0, Ac48, and analogs of eteplirsen anddrisapersen at 5 μM were examined in ex52 del. KM571 cells. Thesuperiority of the oligonucleotides Ac0 and Ac48 of the presentinvention, with respect to exon skipping efficiency and dystrophinprotein rescue, was observed at days 2 and 11 post-transfection comparedto analog AOs of eteplirsen and drisapersen (FIG. 2).

2.5 Dose-Dependent Effects of Ac0, Ac48, and Analog Eteplirsen andDrisapersen

RT-PCR showed that Ac0 at the highest concentration of 10 μM induced upto 74% and 64% exon 51 skipping in DMD KM571 (ex52 del.) and 6594 cells(ex48-50 del.), respectively, which were significantly higher thananalogs of eteplirsen and drisapersen

(FIG. 3). At the lowest concentration (1 μM), Ac0 showed 12 and 10 timeshigher exon skipping efficiency compared to analog eteplirsen in KM571and 6594 cells, respectively. Interestingly, even a concentration of 1μM Ac0 induced higher levels of exon 51 skipping than 10 μM analogeteplirsen (24% efficiency vs 15% in KM571 and 24% efficiency vs 21% in6594, respectively). Quantitative Western blotting revealed that 10 μMAc0 rescued dystrophin protein expression in DMD cell lines up to 21% ofhealthy cell line levels (FIG. 3A to D). Even at 1 μM, the relativeratio of Ac0 to analog eteplirsen represented 7.1 and 3.3 times higherefficiency in producing dystrophin protein in KM571 and 6594 cell lines,respectively. Ac0 at 1 pM enabled the production of rescued dystrophinprotein at higher or comparable levels than analog eteplirsen at 10 μM(10% vs 6% in KM571 and 11% vs 10% in 6594, respectively), confirmingthat Ac0 is more than 10-fold effective at producing dystrophin proteincompared to analog eteplirsen concentration-wise. Analog drisapersen didnot work effectively for either of exon skipping or dystrophinproduction in the DMD muscle cell lines. The exon skipping response toAc0 and Ac48 occurred in a dose-dependent manner that was greater thanboth analog eteplirsen, and analog drisapersen (FIG. 3A and C). Thedose-responsiveness of Ac0 with respect to dystrophin protein productionwas also higher than the control analogs in both DMD cell lines (FIG.3E).

2.6 Immunocytochemical Assessment of Dystrophin Protein Rescue

Immunocytochemistry revealed that Ac0 and Ac48 at 10 μM yielded moredystrophin-positive myotubes and displayed stronger signal intensity inDMD skeletal muscle cell lines harboring ex52 and ex48-50 del. mutationscompared to analog eteplirsen (FIG. 4).

2.7 Length Optimization of Ac0 Morpholino

In silico and in vitro screening revealed that the initial 5′ region ofexon 51 between 0 and +89 is an important region for influencing exon 51skipping. To optimize the sequence length of Ac0 targeting this region,we compared the skipping efficiencies of Ac0 morpholinos of differentlengths (25- to 30-mer), in which nucleotides at the 5′ site weresystematically removed one at a time (see Table 2). In vitro testing inimmortalized DMD muscle cells treated with 1 μM of these AOs showed that25-30-mer Ac0 morpholinos produced efficient exon skipping (>20%) (FIG.5), an effect that was not observed analog eteplirsen, and analogdrisapersen at the same dose (FIG. 3). However, the efficiency of exonskipping increased as the length of the AO was increased. Thestatistically significant effectiveness of 30-mer Ac0 was confirmed at 1and 3 μM doses compared to the shorter Ac0 morpholinos in both celllines, even those AOs that are only 1 or 2 bases shorter.

2.8 Effect of Ac0, Ac48, and Analog Eteplirsen and Drisapersen onPrimary DMD Patient-Derived Skeletal Muscle Cells

We also tested the AOs in primary DMD skeletal muscle cells with exons45-50 (ID 4546) or exons 49-50 del. mutations (ID 4555) to validate ifthe superior efficacy of 30-mer Ac0 is consistent for other muscle celltypes and deletion mutation patterns. RT-PCR showed that Ac0 achievedsignificantly higher exon skipping efficiency in both primary DMD musclecells compared to analog eteplirsen, or analog drisapersen (FIG. 6A toD): up to 5 and 7 times higher efficiency were observed compared toanalog eteplirsen and drisapersen, respectively. A significantefficiency of Ac0-mediated exon 51 skipping was also confirmed inprimary healthy skeletal muscle cells (FIG. 6E and F). Interestingly,with increasing exon 51 skipping efficiency, spontaneous exon 52skipping, which does not disrupt the reading frame, was observed inprimary healthy and DMD muscle cells, and an immortalized DMD musclecell line with ex48-50 del. (6594).

2.10 In Vivo Efficacy of Ac0 Morpholino and Analog Eteplirsen inhDMD/Dmd-Null Mice

A major hurdle in the development of exon skipping therapy is thathuman-specific AOs cannot always be tested in an appropriate animalmodel. This limits the evaluation of in vivo effects of AOs designed forpatients. Here, we developed a new mouse model that has the full-lengthhuman DMD gene but lacks the entire mouse Dmd gene (hDMD/Dmd-null) totest the in vivo efficacy of human AOs. This mouse model was employed toavoid the cross-reaction between human sequences and mouse sequences(note that conventional mdx mice still have the mouse dystrophin mRNA,which can cross-react with human-targeting AOs), and obtained bycross-breeding between hDMD mice34 and Dmd-null mice35. Ac0, Ac48,analog eteplirsen or analog drisapersen was injected into TA muscles ofthese mice, and the effectiveness of in vivo exon 51 skipping wasanalysed 2 weeks after the injection. The result showed significantlygreater exon skipping efficiency in mice treated with Ac0 compared toanalog eteplirsen (FIG. 7). Visible exon 51-skipped bands were found inAc48-treated mice, with an average exon skipping efficiency of 1.11%(±0.46%, SE). On the other hand, no quantifiable exon 51-skipped bandswere observed in mice treated with analog drisapersen (FIG. 8).

Sequences Ac0 (SEQ ID NO. 1) GTGTCACCAGAGTAACAGTCTGAGTAGGAG Ac5(SEQ ID NO. 2) AGGTTGTGTCACCAGAGTAACAGTCTGAGT Ac26 (SEQ ID NO. 3)GGCAGTTTCCTTAGTAACCACAGGTTGTGT Ac30 (SEQ ID NO. 4)AGATGGCAGTTTCCTTAGTAACCACAGGTT Ac48 (SEQ ID NO. 5)ATGGCATTTCTAGTTTGGAGATGGCAGTTT Eteplirsen (SEQ ID NO. 6)CTCCAACATCAAGGAAGATGGCATTTCTAG Drisapersen (SEQ ID NO. 7)TCAAGGAAGATGGCATTTCT hEx51_Ac0-29mer (SEQ ID NO. 8)TGTCACCAGAGTAACAGTCTGAGTAGGAG hEx51_Ac0-28mer (SEQ ID NO. 9)GTCACCAGAGTAACAGTCTGAGTAGGAG hEx51_Ac0-27mer (SEQ ID NO. 10)TCACCAGAGTAACAGTCTGAGTAGGAG hEx51_Ac0-26mer (SEQ ID NO. 11)CACCAGAGTAACAGTCTGAGTAGGAG hEx51_Ac0-25mer (SEQ ID NO. 12)ACCAGAGTAACAGTCTGAGTAGGAG Ac9 (SEQ ID NO. 13)CCACAGGTTGTGTCACCAGAGTAACAGTCT Ac141 (SEQ ID NO. 14)TTATAACTTGATCAAGCAGAGAAAGCCAGT Ac207 (SEQ ID NO. 15)atacCTTCTGCTTGATGATCATCTCGTTGA Ex49/50_94-10_hDMD_Fwd (SEQ ID NO. 16)CAGCCAGTGAAGAGGAAGTTAG Ex53_80-99_hDMD_Rv (SEQ ID NO. 17)CCAGCCATTGTGTTGAATCC Ex47_60-79_hDMD_Fwd (SEQ ID NO. 18)AGGACCCGTGCTTGTAAGTG Ex52_83-105_hDMD_Rv (SEQ ID NO. 19)GATTGTTCTAGCCTCTTGATTGC Ex43/44_167-12_hDMD_Fwd (SEQ ID NO. 20)GACAAGGGCGATTTGACAG mDmd_ex49_83-102_Fwd (SEQ ID NO. 21)CAAGCACTCAGCCAGTGAAG deletion mDmd_ex53_81-100_Rv (SEQ ID NO. 22)TCCAGCCATTGTGTTGAATC hGAPDH_662-81_Fwd (SEQ ID NO. 23)TCCCTGAGCTGAACGGGAAG control hGAPDH_860-79_Rv (SEQ ID NO. 24)TCCAGCCATTGTGTTGAATC h185_760-82_Fwd (SEQ ID NO. 25)TCGATGCTCTTAGCTGAGTGTCC control h18S_1039-58_Rv (SEQ ID NO. 26)TGATCGTCTTCGAACCTCCG

TABLE 3 51 9 CCACAGGTTGTGTCACCAGAGTAACAGTCT 80.49 1 18GTAACCACAGGTTGTGTCACCAGAG 41.20 1 51 0 GTGTCACCAGAGTAACAGTCTGAGTAGGAG80.11 2 16 AACCACAGGTTGTGTCACCAGAGTA 39.94 2 51 10ACCACAGGTTGTGTCACCAGAGTAACAGTC 79.98 3 12 ACAGGTTGTGTCACCAGAGTAACAG38.08 3 51 5 AGGTTGTGTCACCAGAGTAACAGTCTGAGT 72.97 4 14CCACAGGTTGTGTCACCAGAGTAAC 37.52 4 51 8 CACAGGTTGTGTCACCAGAGTAACAGTCTG72.01 5 15 ACCACAGGTTGTGTCACCAGAGTAA 37.23 5 51 1TGTGTCACCAGAGTAACAGTCTGAGTAGGA 71.94 6 31 GGCAGTTTCCTTAGTAACCACAGGT37.18 6 51 2 TTGTGTCACCAGAGTAACAGTCTGAGTAGG 71.51 7 13CACAGGTTGTGTCACCAGAGTAACA 36.66 7 51 11 AACCACAGGTTGTGTCACCAGAGTAACAGT70.65 8 10 AGGTTGTGTCACCAGAGTAACAGTC 35.56 8 51 6CAGGTTGTGTCACCAGAGTAACAGTCTGAG 68.18 9 11 CAGGTTGTGTCACCAGAGTAACAGT33.75 9 51 7 ACAGGTTGTGTCACCAGAGTAACAGTCTGA 68.14 10 0ACCAGAGTAACAGTCTGAGTAGGAG 33.34 10 51 4 GGTTGTGTCACCAGAGTAACAGTCTGAGTA66.65 11 9 GGTTGTGTCACCAGAGTAACAGTCT 33.10 11 51 26GGCAGTTTCCTTAGTAACCACAGGTTGTGT 66.32 12 17 TAACCACAGGTTGTGTCACCAGAGT32.95 12 51 18 CCTTAGTAACCACAGGTTGTGTCACCAGAG 65.25 13 32TGGCAGTTTCCTTAGTAACCACAGG 32.77 13 51 19 TCCTTAGTAACCACAGGTTGTGTCACCAGA64.81 14 30 GCAGTTTCCTTAGTAACCACAGGTT 31.61 14 51 27TGGCAGTTTCCTTAGTAACCACAGGTTGTG 64.09 15 19 AGTAACCACAGGTTGTGTCACCAGA30.95 15 51 12 TAACCACAGGTTGTGTCACCAGAGTAACAG 64.08 16 23CCTTAGTAACCACAGGTTGTGTCAC 30.66 16 51 13 GTAACCACAGGTTGTGTCACCAGAGTAACA63.65 17 5 GTGTCACCAGAGTAACAGTCTGAGT 30.54 17 51 25GCAGTTTCCTTAGTAACCACAGGTTGTGTC 61.81 18 1 CACCAGAGTAACAGTCTGAGTAGGA30.26 18 51 29 GATGGCAGTTTCCTTAGTAACCACAGGTTG 61.44 19 6TGTGTCACCAGAGTAACAGTCTGAG 29.52 19 51 14 AGTAACCACAGGTTGTGTCACCAGAGTAAC57.56 20 24 TCCTTAGTAACCACAGGTTGTGTCA 28.97 20 51 23AGTTTCCTTAGTAACCACAGGTTGTGTCAC 57.29 21 2 TCACCAGAGTAACAGTCTGAGTAGG28.83 21 51 3 GTTGTGTCACCAGAGTAACAGTCTGAGTAG 56.65 22 3GTCACCAGAGTAACAGTCTGAGTAG 26.60 22 51 17 CTTAGTAACCACAGGTTGTGTCACCAGAGT56.39 23 25 TTCCTTAGTAACCACAGGTTGTGTC 26.23 23 51 24CAGTTTCCTTAGTAACCACAGGTTGTGTCA 56.16 24 27 GTTTCCTTAGTAACCACAGGTTGTG26.11 24 51 30 AGATGGCAGTTTCCTTAGTAACCACAGGTT 55.46 25 29CAGTTTCCTTAGTAACCACAGGTTG 24.24 25 51 20 TTCCTTAGTAACCACAGGTTGTGTCACCAG53.39 26 34 GATGGCAGTTTCCTTAGTAACCACA 22.08 26 51 16TTAGTAACCACAGGTTGTGTCACCAGAGTA 53.04 27 8 GTTGTGTCACCAGAGTAACAGTCTG21.25 27 51 15 TAGTAACCACAGGTTGTGTCACCAGAGTAA 52.28 28 7TTGTGTCACCAGAGTAACAGTCTGA 21.05 28 51 22 GTTTCCTTAGTAACCACAGGTTGTGTCACC51.90 29 4 TGTCACCAGAGTAACAGTCTGAGTA 20.02 29 51 28ATGGCAGTTTCCTTAGTAACCACAGGTTGT 46.50 30 26 TTTCCTTAGTAACCACAGGTTGTGT19.62 30 51 21 TTTCCTTAGTAACCACAGGTTGTGTCACCA 45.73 31 22CTTAGTAACCACAGGTTGTGTCACC 18.61 31 51 31 GAGATGGCAGTTTCCTTAGTAACCACAGGT43.71 32 33 ATGGCAGTTTCCTTAGTAACCACAG 18.23 32 51 32GGAGATGGCAGTTTCCTTAGTAACCACAGG 38.58 33 108 TCTGTCCAAGCCCGGTTGAAATCTG18.06 33 51 98 CCAAGCCCGGTTGAAATCTGCCAGAGCAGG 36.79 34 87TCTGCCAGAGCAGGTACCTCCAACA 18.04 34 51 77 CAGAGCAGGTACCTCCAACATCAAGGAAGA36.22 35 98 CCCGGTTGAAATCTGCCAGAGCAGG 17.52 35 51 46GGCATTTCTAGTTTGGAGATGGCAGTTTCC 35.05 36 28 AGTTTCCTTAGTAACCACAGGTTGT16.78 36 51 102 CTGTCCAAGCCCGGTTGAAATCTGCCAGAG 33.96 37 35AGATGGCAGTTTCCTTAGTAACCAC 16.49 37 51 103 TCTGTCCAAGCCCGGTTGAAATCTGCCAGA33.85 38 20 TAGTAACCACAGGTTGTGTCACCAG 16.42 38 51 78CCAGAGCAGGTACCTCCAACATCAAGGAAG 32.83 39 83 CCAGAGCAGGTACCTCCAACATCAA15.89 39 51 100 GTCCAAGCCCGGTTGAAATCTGCCAGAGCA 32.12 40 86CTGCCAGAGCAGGTACCTCCAACAT 15.09 40 51 101 TGTCCAAGCCCGGTTGAAATCTGCCAGAGC31.85 41 82 CAGAGCAGGTACCTCCAACATCAAG 14.95 41 51 53GGAAGATGGCATTTCTAGTTTGGAGATGGC 31.58 42 84 GCCAGAGCAGGTACCTCCAACATCA14.91 42 51 99 TCCAAGCCCGGTTGAAATCTGCCAGAGCAG 31.36 43 85TGCCAGAGCAGGTACCTCCAACATC 14.66 43 51 106 AGTTCTGTCCAAGCCCGGTTGAAATCTGCC31.20 44 110 GTTCTGTCCAAGCCCGGTTGAAATC 13.87 44 51 33TGGAGATGGCAGTTTCCTTAGTAACCACAG 30.45 45 107 CTGTCCAAGCCCGGTTGAAATCTGC12.76 45 51 105 GTTCTGTCCAAGCCCGGTTGAAATCTGCCA 29.76 46 109TTCTGTCCAAGCCCGGTTGAAATCT 12.71 46 51 104 TTCTGTCCAAGCCCGGTTGAAATCTGCCAG29.45 47 99 GCCCGGTTGAAATCTGCCAGAGCAG 12.07 47 51 117GCCAGTCGGTAAGTTCTGTCCAAGCCCGGT 28.99 48 81 AGAGCAGGTACCTCCAACATCAAGG11.09 48 51 87 TGAAATCTGCCAGAGCAGGTACCTCCAACA 27.92 49 36GAGATGGCAGTTTCCTTAGTAACCA 10.96 49 51 37 AGTTTGGAGATGGCAGTTTCCTTAGTAACC27.37 50 51 GGCATTTCTAGTTTGGAGATGGCAG 10.71 50 51 97CAAGCCCGGTTGAAATCTGCCAGAGCAGGT 27.25 51 111 AGTTCTGTCCAAGCCCGGTTGAAAT10.66 51 51 40 TCTAGTTTGGAGATGGCAGTTTCCTTAGTA 27.24 52 21TTAGTAACCACAGGTTGTGTCACCA 10.43 52 51 76 AGAGCAGGTACCTCCAACATCAAGGAAGAT27.06 53 106 TGTCCAAGCCCGGTTGAAATCTGCC 10.38 53 51 81CTGCCAGAGCAGGTACCTCCAACATCAAGG 26.57 54 112 AAGTTCTGTCCAAGCCCGGTTGAAA9.48 54 51 95 AGCCCGGTTGAAATCTGCCAGAGCAGGTAC 26.37 55 115GGTAAGTTCTGTCCAAGCCCGGTTG 9.33 55 51 86 GAAATCTGCCAGAGCAGGTACCTCCAACAT25.98 56 50 GCATTTCTAGTTTGGAGATGGCAGT 9.05 56 51 80TGCCAGAGCAGGTACCTCCAACATCAAGGA 25.94 57 101 AAGCCCGGTTGAAATCTGCCAGAGC8.92 57 51 96 AAGCCCGGTTGAAATCTGCCAGAGCAGGTA 25.55 58 103CCAAGCCCGGTTGAAATCTGGCAGA 8.24 58 51 79 GCCAGAGCAGGTACCTCCAACATCAAGGAA25.55 59 113 TAAGTTCTGTCCAAGCCCGGTTGAA 7.96 59 51 108TAAGTTCTGTCCAAGCCCGGTTGAAATCTG 25.54 60 105 GTCCAAGCCCGGTTGAAATCTGCCA7.95 60 51 90 GGTTGAAATCTGCCAGAGCAGGTACCTCCA 25.44 61 53ATGGCATTTCTAGTTTGGAGATGGC 7.67 61 51 50 AGATGGCATTTCTAGTTTGGAGATGGCAGT25.21 62 100 AGCCCGGTTGAAATCTGCCAGAGCA 7.33 62 51 89GTTGAAATCTGCCAGAGCACGTACCTCCAA 24.68 63 116 CGGTAAGTTCTGTCCAAGCCCGGTT7.17 63 51 94 GCCCGGTTGAAATCTGCCAGAGCAGGTACC 23.96 64 97CCGGTTGAAATCTGCCAGAGCAGGT 6.96 64 51 88 TTGAAATCTGCCAGAGCAGCTACCTCCAAC23.90 65 117 TCGGTAAGTTCTGTCCAAGCCCGGT 6.90 65 51 34TTGGACATGCCACTTTCCTTAGTAACCACA 23.77 66 102 CAAGCCCGGTTGAAATCTGCCAGAG6.69 66 51 65 CTCCAACATCAAGGAAGATGGCATTTCTAG 23.66 67 114GTAAGTTCTGTCCAAGCCCGGTTGA 6.65 67 51 35 TTTGGAGATGGCAGTTTCCTTAGTAACCAC23.53 68 104 TCCAAGGCCGGTTGAAATCTGCCAG 6.63 68 51 91CGGTTGAAATCTGCCAGAGCAGGTACCTCC 23.52 69 91 GAAATCTGCCAGAGCAGGTACCTCC6.24 69 51 45 GCATTTCTAGTTTGGAGATGGCAGTTTCCT 23.40 70 37GGAGATGGCAGTTTCCTTAGTAACC 6.17 70 51 57 TCAAGGAAGATGGCATTTCTAGTTTGCAGA23.34 71 88 ATCTGCCAGAGCAGGTACCTCCAAC 6.14 71 51 118AGCCAGTCGGTAAGTTCTGTCCAAGCCCGG 23.29 72 118 GTCGGTAAGTTCTGTCCAAGCCCGG5.97 72 51 82 TCTGCCAGAGCAGGTACCTCCAACATCAAG 22.86 73 95GGTTGAAATCTGCCAGAGCAGGTAC 5.25 73 51 93 CCCGGTTGAAATCTGCCAGAGCAGGTACCT22.81 74 57 GAAGATGGCATTTCTAGTTTGGAGA 5.02 74 51 107AAGTTCTGTCCAAGCCCGGTTGAAATCTGC 22.77 75 46 TTCTAGTTTGGAGATGGCAGTTTCC5.00 75 51 116 CCAGTCGGTAAGTTCTGTCCAAGCCCGGTT 22.52 76 47TTTCTAGTTTGGAGATGGCAGTTTC 4.87 76 51 109 GTAAGTTCTGTCCAAGCCCGGTTGAAATCT22.51 77 89 AATCTGCCAGAGCAGGTACCTCCAA 4.60 77 51 110GGTAAGTTCTGTCCAAGCCCGGTTGAAATC 22.46 78 58 GGAAGATGGCATTTCTAGTTTGGAG4.30 78 51 92 CCGGTTGAAATCTGCCAGAGCAGGTACCTC 22.07 79 96CGGTTGAAATCTGCCAGAGCAGGTA 3.79 79 51 36 GTTTGGAGATGGCAGTTTCCTTAGTAACCA21.97 80 131 GCAGAGAAAGCCAGTCGGTAAGTTC 3.63 80 51 56CAAGGAAGATGGCATTTCTAGTTTGGAGAT 21.85 81 80 GAGCAGGTACCTCCAACATCAAGGA3.51 81 51 75 GAGCAGGTACCTCCAACATCAAGGAAGATG 21.81 82 120CAGTCGGTAAGTTCTGTCCAAGCCC 3.35 82 51 39 CTAGTTTGGAGATGGCAGTTTCCTTAGTAA21.62 83 128 GAGAAAGCCAGTCGGTAAGTTCTGT 3.12 83 51 64TCCAACATCAAGGAAGATGGCATTTCTAGT 21.18 84 119 AGTCGGTAAGTTCTGTCCAAGCCCG3.09 84 51 115 CAGTCGGTAAGTTCTGTCCAAGCCCGGTTG 20.90 85 92TGAAATCTGCCAGAGCAGGTACCTC 2.10 85 51 52 GAAGATGGCATTTCTAGTTTGGAGATGGCA20.77 86 40 TTTGGAGATGGCAGTTTCCTTAGTA 1.87 86 51 63CCAACATCAAGGAAGATGGCATTTCTAGTT 20.61 87 90 AAATCTGCCAGAGCAGGTACCTCCA1.74 87 51 111 CGGTAAGTTCTGTCCAAGCCCGGTTGAAAT 20.60 88 129AGAGAAAGCCAGTCGGTAAGTTCTG 1.57 88 51 41 TTCTAGTTTGGAGATGGCAGTTTCCTTAGT20.19 89 121 CCAGTCGGTAAGTTCTGTCCAAGCC 1.52 89 51 84AATCTGCCAGAGCAGGTACCTCCAACATCA 19.86 90 122 GCCAGTCGGTAAGTTCTGTCCAAGC0.99 90 51 113 GTCGGTAAGTTCTGTCCAAGCCCGGTTGAA 19.80 91 38TGGAGATGGCAGTTTCCTTAGTAAC 0.44 91 51 114 AGTCGGTAAGTTCTGTCCAAGCCCGGTTGA19.75 92 45 TCTAGTTTGGAGATGGCAGTTTCCT 0.40 92 51 73GCAGGTACCTCCAACATCAAGGAAGATGGC 19.30 93 78 GCAGGTACCTCCAACATCAAGGAAG0.10 93 51 38 TAGTTTGGAGATGGCAGTTTCCTTAGTAAC 19.21 94 48ATTTCTAGTTTGGAGATGGCAGTTT −0.21 94 51 119 AAGCCAGTCGGTAAGTTCTGTCCAAGCCCG19.04 95 125 AAAGCCAGTCGGTAAGTTCTGTCCA −0.23 95 51 67ACCTCCAACATCAAGGAAGATGGCATTTCT 19.03 96 126 GAAAGCCAGTCGGTAAGTTCTGTCC−0.58 96 51 83 ATCTGCCAGAGCAGGTACCTCCAACATCAA 18.98 97 39TTGGAGATGGCAGTTTCCTTAGTAA −0.64 97 51 58 ATCAAGGAAGATGGCATTTCTAGTTTGGAG18.74 98 134 CAAGCAGAGAAAGCCAGTCGGTAAG −0.80 98 51 112TCGGTAAGTTCTGTCCAAGCCCGGTTGAAA 18.59 99 132 AGCAGAGAAAGCCAGTCGGTAAGTT−0.92 99 51 54 AGGAAGATGGCATTTCTAGTTTGGAGATGG 18.20 100 137GATCAAGCAGAGAAAGCCAGTCGGT −0.94 100 51 85 AAATCTGCCAGAGCAGGTACCTCCAACATC17.93 101 133 AAGCAGAGAAAGCCAGTCGGTAAGT −1.00 101 51 66CCTCCAACATCAAGGAAGATGGCATTTCTA 17.65 102 138 TGATCAAGCAGAGAAAGCCAGTCGG−1.03 102 51 49 GATGGCATTTCTAGTTTGGAGATGGCAGTT 17.50 103 127AGAAAGCCAGTCGGTAAGTTCTGTC −1.07 103 51 42 TTTCTAGTTTGGAGATGGCAGTTTCCTTAG17.40 104 136 ATCAAGCAGAGAAAGCCAGTCGGTA −1.36 104 51 44CATTTCTAGTTTGGAGATGGCAGTTTCCTT 16.99 105 72 ACCTCCAACATCAAGGAAGATGGCA−1.50 105 51 51 AAGATGGCATTTCTAGTTTGGAGATGGCAG 16.77 106 79AGCAGGTACCTCCAACATCAAGGAA −1.50 106 51 55 AAGGAAGATGGCATTTCTAGTTTGGAGATG16.50 107 130 CAGAGAAAGCCAGTCGGTAAGTTCT −1.79 107 51 68TACCTCCAACATCAAGGAAGATGGCATTTC 16.36 108 94 GTTGAAATCTGCCAGAGCAGGTACC−1.88 108 51 121 GAAAGCCAGTCGGTAAGTTCTGTCCAAGCC 16.27 109 59AGGAAGATGGCATTTCTAGTTTGGA −2.14 109 51 120AAAGCCAGTCGGTAAGTTCTGTCCAAGCCC 16.02 110 56 AAGATGGCATTTCTAGTTTGGAGAT−2.16 110 51 74 AGCAGGTACCTCCAACATCAAGGAAGATGG 15.45 111 93TTGAAATCTGCCAGAGCAGGTACCT −2.19 111 51 122AGAAAGCCAGTCGGTAAGTTCTGTCCAAGC 15.40 112 140 CTTGATCAAGCAGAGAAAGCCAGTC−2.58 112 51 59 CATCAAGGAAGATGGCATTTCTAGTTTGGA 15.35 113 70CTCCAACATCAAGGAAGATGGCATT −2.88 113 51 123GAGAAAGCCAGTCGGTAAGTTCTGTCCAAG 15.01 114 71 CCTCCAACATCAAGGAAGATGGCAT−2.97 114 51 69 GTACCTCCAACATCAAGGAAGATGGCATTT 14.79 115 52TGGCATTTCTAGTTTGGAGATGGCA −3.00 115 51 60 ACATCAAGGAAGATGGCATTTCTAGTTTGG14.56 116 63 ATCAAGGAAGATGGCATTTCTAGTT −3.06 116 51 72CAGGTACCTCCAACATCAAGGAAGATGGCA 13.59 117 42 AGTTTGGAGATGGCAGTTTCCTTAG−3.16 117 51 124 AGAGAAAGCCAGTCGGTAAGTTCTGTCCAA 12.92 118 54GATGGCATTTCTAGTTTGGAGATGG −3.16 118 51 70 GGTACCTCCAACATCAAGGAAGATGGCATT12.45 119 49 CATTTCTAGTTTGGAGATGGCAGTT −3.37 119 51 71AGGTACCTCCAACATCAAGGAAGATGGCAT 12.24 120 135 TCAAGCAGAGAAAGCCAGTCGGTAA−3.39 120 51 62 CAACATCAAGGAAGATGGCATTTCTAGTTT 12.18 121 123AGCCAGTCGGTAAGTTCTGTCCAAG −3.44 121 51 61 AACATCAAGGAAGATGGCATTTCTAGTTTG12.09 122 141 ACTTGATCAAGCAGAGAAAGCCAGT −3.99 122 51 126GCAGAGAAAGCCAGTCGGTAAGTTCTGTCC 12.04 123 68 CCAACATCAAGGAAGATGGCATTTC−4.03 123 51 125 CAGAGAAAGCCAGTCGGTAAGTTCTGTCCA 11.49 124 124AAGCCAGTCGGTAAGTTCTGTCCAA −4.20 124 51 43 ATTTCTAGTTTGGAGATGGCAGTTTCCTTA11.13 125 60 AAGGAAGATGGCATTTCTAGTTTGG −4.22 125 51 47TGGCATTTCTAGTTTGGAGATGGCAGTTTC 11.09 126 55 AGATGGCATTTCTAGTTTGGAGATG−4.42 126 51 129 CAAGCAGAGAAAGCCAGTCGGTAAGTTCTG 10.80 127 142AACTTGATCAAGCAGAGAAAGCCAG −4.53 127 51 48 ATGGCATTTCTAGTTTGGAGATGGCAGTTT10.61 128 73 TACCTCCAACATCAAGGAAGATGGC −4.70 128 51 130TCAAGCAGAGAAAGCCAGTCGGTAAGTTCT 10.43 129 139 TTGATCAAGCAGAGAAAGCCAGTCG−4.97 129 51 128 AAGCAGAGAAAGCCAGTCGGTAAGTTCTGT 9.84 130 41GTTTGGAGATGGCAGTTTCCTTAGT −5.02 130 51 131ATCAAGCAGAGAAAGCCAGTCGGTAAGTTC 8.53 131 74 GTACCTCCAACATCAAGGAAGATGG−5.30 131 51 127 AGCAGAGAAAGCCAGTCGGTAAGTTCTGTC 8.26 132 64CATCAAGGAAGATGGCATTTCTAGT −5.36 132 51 136ACTTGATCAAGCAGAGAAAGCCAGTCGGTA 6.43 133 65 ACATCAAGGAAGATGGCATTTCTAG−5.43 133 51 137 AACTTGATCAAGCAGAGAAAGCCAGTCGGT 5.84 134 44CTAGTTTGGAGATGGCAGTTTCCTT −6.16 134 51 138TAACTTGATCAAGCAGAGAAAGCCAGTCGG 5.26 135 61 CAAGGAAGATGGCATTTCTAGTTTG−6.27 135 51 132 GATCAAGCAGAGAAAGCCAGTCGGTAAGTT 4.60 136 172GTCACCCACCATCACCCTCTGTGAT −7.68 136 51 140TATAACTTGATCAACCAGAGAAAGCCAGTC 4.36 137 69 TCCAACATCAAGGAAGATGGCATTT−7.81 137 51 133 TGATCAAGCAGAGAAAGCCAGTCGGTAAGT 4.31 138 143TAACTTGATCAAGCAGAGAAAGCCA −8.29 138 51 134TTGATCAAGCAGAGAAAGCCAGTCGGTAAG 3.75 139 62 TCAAGGAAGATGGCATTTCTAGTTT−8.30 139 51 139 ATAACTTGATCAAGCAGAGAAAGCCAGTCG 2.70 140 77CAGGTACCTCCAACATCAAGGAAGA −8.53 140 51 135CTTGATCAAGCAGAGAAAGCCAGTCGGTAA 2.28 141 67 CAACATCAAGGAAGATGGCATTTCT−8.81 141 51 141 TTATAACTTGATCAAGCAGAGAAAGCCAGT 1.76 142 173GGTCACCCACCATCACCCTCTGTGA −8.87 142 51 142TTTATAACTTGATCAAGCAGAGAAAGCCAG −0.17 143 144 ATAACTTGATCAAGCAGAGAAAGCC−8.98 143 51 146 TGATTTTATAACTTGATCAAGCAGAGAAAG −3.06 144 75GGTACCTCCAACATCAAGGAAGATG −9.18 144 51 145GATTTTATAACTTGATCAAGCAGAGAAAGC −3.17 145 174 AGGTCACCCACCATCACCCTCTGTG−9.52 145 51 156 TCACCCTCTGTGATTTTATAACTTGATCAA −3.49 146 66AACATCAAGGAAGATGGCATTTCTA −9.58 146 51 167GTCACCCACCATCACCCTCTGTGATTTTAT −3.97 147 43 TAGTTTGGAGATGGCAGTTTCCTTA−9.61 147 51 143 TTTTATAACTTGATCAAGCAGAGAAAGCCA −4.01 148 146TTATAACTTGATCAAGCAGAGAAAG −9.74 148 51 174CCTCAAGGTCACCCACCATCACCCTCTGTG −4.37 149 171 TCACCCACCATCACCCTCTGTGATT−10.00 149 51 144 ATTTTATAACTTGATCAAGCAGAGAAAGCC −4.66 150 170CACCCACCATCACCCTCTGTGATTT −10.19 150 51 155CACCCTCTGTGATTTTATAACTTGATCAAG −5.42 151 147 TTTATAACTTGATCAAGCAGAGAAA−10.27 151 51 168 GGTCACCCACCATCACCCTCTGTGATTTTA −5.53 152 176CAAGGTCACCCACCATCACCCTCTG −11.06 152 51 175TCCTCAAGGTCACCCACCATCACCCTCTGT −5.64 153 145 TATAACTTGATCAAGCAGAGAAAGC−11.20 153 51 157 ATCACCCTCTGTGATTTTATAACTTGATCA −5.75 151 168CCCACCATCACCCTCTGTGATTTTA −11.23 151 51 173CTCAAGGTCACCCACCATCACCCTCTGTGA −6.14 155 169 ACCCACCATCACCCTCTGTGATTTT−11.29 155 51 176 ATCCTCAAGGTCACCCACCATCACCCTCTG −6.36 156 177TCAAGGTCACCCACCATCACCCTCT −11.45 156 51 172TCAAGGTCACCCACCATCACCCTCTGTGAT −6.44 157 181 ATCCTCAAGGTCACCCACCATCACC−11.30 157 51 166 TCACCCACCATCACCCTCTGTGATTTTATA −6.45 158 175AAGGTCACCCACCATCACCCTCTGT −11.35 158 51 163CCCACCATCACCCTCTGTGATTTTATAACT −6.75 159 180 TCCTCAAGGTCACCCACCATCACCC−11.59 159 51 161 CACCATCACCCTCTGTGATTTTATAACTTG −6.93 160 179CCTCAAGCTCACCCACCATCACCCT −11.76 160 51 147GTGATTTTATAACTTGATCAAGCAGAGAAA −7.04 161 183 ATATCCTCAAGGTCACCCACCATCA−11.76 161 51 164 ACCCACCATCACCCTCTGTGATTTTATAAC −7.04 162 178CTCAAGGTCACCCACCATCACCCTC −11.79 162 51 171CAAGGTCACCCACCATCACCCTCTGTGATT −7.07 163 76 AGGTACCTCCAACATCAAGGAAGAT−12.00 163 51 169 AGGTCACCCACCArCACCCTCTGTGAJTTT −7.08 161 182TATCCTCAAGGTCACCCACCATCAC −12.19 164 51 153CCCTCTGTGATTTTATAACTTGATCAAGCA −7.09 165 156 CTCTGTGATTTTATAACTTGATCAA−12.23 165 51 165 CACCCACCATCACCCTCTGTGATTTTATAA −7.12 166 167CCACCATCACCCTCTGTGATTTTAT −12.52 166 51 178ATATCCTCAAGGTCACCCACCATCACCCTC −7.12 167 157 CCTCTGTGATTTTATAACTTGATCA−12.99 167 51 160 ACCATCACCCTCTGTG.MTTTATAACTTGA −7.13 168 184GATATCCTCAAGGTCACCCACCATC −13.02 168 51 177TATCCTCAAGGTCACCCACCATCACCCTCT −7.18 169 148 TTTTATAACTTGATCAAGCAGAGAA−13.20 169 51 154 ACCCTCTGTGATTTTATAACTTGATCAAGC −7.53 170 190CTCGTTGATATCCTCAAGGTCACCC −13.32 170 51 170AAGGTCACCCACCATCACCCTCTGTGATTT −7.89 171 189 TCGTTGATATCCTCAAGGTCACCCA−13.68 171 51 162 CCACCATCACCCTCTGTGATTTTATAACTT −8.37 172 185TGATATCCTCAAGGTCACCCACCAT −13.68 172 51 159CCATCACCCTCTGTGATTTTATAACTTGAT −8.43 173 188 CGTTGATATCCTCAAGGTCACCCAC−13.80 173 51 158 CATCACCCTCTGTGATTTTArAACTTGATC −8.95 174 149ATTTTATAACTTGATCAAGCAGAGA −13.86 174 51 179GATATCCTCAAGGTCACCCACCATCACCCT −9.14 175 191 TCTCGTTGATATCCTCAAGGTCACC−13.88 175 51 152 CCTCTGTGATTTTATAACTTGATCAAGCAG −9.35 176 160CACCCTCTGTGATTTTATAACTTGA −13.94 176 51 149CTGTGATTITATAACTTGATCAAGCAGAGA −9.51 177 187 GTTGATATCCTCAAGGTCAGCCACC−14.07 177 51 180 TGATATCCTCAAGGTCACCCACCATCACCC −9.82 178 155TCTGTGATTTTATAACTTGATCAAG −14.17 178 51 181TTGATATCCTCAAGGTCACCCACCATCACC −9.89 179 158 CCCTCTGTGATTTTATAACTTGATC−14.23 179 51 148 TGTGATTTTATAACTTGATCAAGCAGAGAA −10.04 180 192ATCTCGTTGATATCCTCAAGGTCAC −14.36 180 51 182GTTGATATCCTCAAGGTCACCCACCATCAC −10.09 181 186 TTGATATCCTCAAGGTCACCCACCA−14.42 181 51 183 CGTTGATATCCTCAAGGTCACCCACCATCA −10.50 182 193CATCTCGTTGATATCCTCAAGGTCA −14.55 182 51 184TCGTTGATATCCTCAAGGTCACCCACCATC −10.67 183 161 TCACCCTCTGTGATTTTATAACTTG−14.60 183 51 185 CTCGTTGATATCCrCAAGGTCACCCACCAT −10.79 184 166CACCATCACCCTCTGTGATTTTATA −14.64 184 51 186TCTCGTTGATATCCTCAAGGTCACCCACCA −11.32 185 194 TCATCTCGTTGATATCCTCAAGGTC−14.73 185 51 188 CATCTCGTTGATATCCTCAAGGTCACCCAC −11.44 186 196GATCATCTCGTTGATATCCTCAAGG −14.81 186 51 189TCATCTCGTTGATATCCTCAAGGTCACCCA −11.81 187 150 GATTTTATAACTTGATCAAGCAGAG−14.89 187 51 187 ATCTCGTTGATATGCTCAAGGTCACCCACC −11.82 188 197TGATCATCTCGTTGATATCCTCAAG −14.91 188 51 190ATCATCTCGTTGATATCCTCAAGGTCACCC −12.10 189 199 GATGATCATCTCGTTGATATCCTCA−14.93 189 51 191 GATCATCTCGTTGATATCCTCAAGGTCACC −12.59 190 198ATGATCATCTCGTTGATATCCTCAA −14.94 190 51 151CTCTGTGATTTTATAACTTGATCAAGCAGA −12.71 191 200 TGATGATCATCTCGTTGATATCCTC−14.98 191 51 192 TGATCATCTCGTTGATATCCTCAAGGTCAC −13.26 192 201TTGATGATCATCTCGTTGATATCCT −15.00 192 51 150TCTGTGATTTTATAACTTGATCAAGCAGAG −13.47 193 195 ATCATCTCGTTGATATCCTCAAGGT−15.11 193 51 193 ATGATCATCTCGTTGATATCCTCAAGGTCA −13.85 194 202CTTGATGATCATCTCGTTGATATCC −15.16 194 51 194GATGATCATCTCGTTGATATCCTCAAGGTC −13.95 195 159 ACCCTCTGTGATTTTATAACTTGAT−15.57 195 51 195 TGATGATCATCTCGTTGATATCCTCAAGGT −14.35 196 201TGCTTGATGATCATCTCGTTGATAT −15.61 196 51 196TTGATGATCATCTCGTTGATATCCTCAAGG −14.51 197 203 GCTTGATGATCATCTCGTTGATATC−15.64 197 51 198 GCTTGATGATCArCTCGTTGATATCCTCAA −14.38 198 164CCArCACCCTCTGTGATTTTATAAC −15.79 198 51 197CTTGATGATCATCTCGTTGATATCCTCAAG −14.70 199 205 CTGCTTGATGATCATCTCGTTGATA−16.21 199 51 199 TGCTTGATGATCATCTCGTTGATATCCTCA −14.77 200 165ACCATCACCCTCTGTGATTTTATAA −16.41 200 51 200CTGCTTGATGATCATCTCGTTGATATCCTC −15.02 201 163 CATCACCCTCTGTGATTTTATAACT−16.49 201 51 201 TCTGCTTGATGATCATCTCGTTGATATCCT −15.29 202 206TCTGCTTGATGATCATCTCGTTGAT −16.57 202 51 202TTCTGCTTGATGATCATCTCGTTGATATCC −15.67 203 207 TTCTGCTrGATGATCATCTCGTTGA−16.92 203 51 203 CTTCTGCTTGATGATCATCTCGTTGATATC −16.24 204 208CTTCTGCTTGATGATCATCTCGTTG −17.32 204 51 154 CTGTGATTTTATAACTTGATCAAGC−17.42 205 51 162 ATCACCCTCTGTGATTTTATAACTT −17.53 206 51 152GTGATTTTATAACTTGATCAAGCAG −17.91 207 51 151 TGATTTTATAACTTGATCAAGCAGA−17.98 208 51 153 TGTGATTTTATAACTTGATCAAGCA −19.57 209

The invention claimed is:
 1. A morpholino antisense oligonucleotide(PMO) consisting of SEQ ID NO:1 (Ac0), SEQ ID NO:2 (Ac5), SEQ ID NO:3(Ac26), SEQ ID NO:4 (Ac30), or SEQ ID NO:5 (Ac48), wherein the PMO bindsto the human dystrophin pre-mRNA of exon 51 within the region between 0and +89 of the pre-mRNA sequence and induces exon skipping of exon 51 ofthe human dystrophin pre-mRNA.
 2. A conjugate comprising the morpholinoantisense oligonucleotide according to claim 1 and a carrier, whereinthe carrier is conjugated to the morpholino antisense oligonucleotide.3. A conjugate according to claim 2, wherein the carrier is operable totransport the antisense oligonucleotide into a target cell.
 4. Aconjugate according to claim 2, wherein the carrier is selected from apeptide, a small molecule chemical, a polymer, a nanoparticle, a lipid,a liposome or an exosome.
 5. A conjugate according to claim 2 whereinthe carrier is a cell penetrating peptide.
 6. A conjugate according toclaim 2 wherein the carrier is an arginine-rich cell penetratingpeptide.
 7. A method of treating a muscular disorder in a subject, themethod comprising administering the morpholino antisense oligonucleotideaccording to claim 1 to a subject in need thereof.
 8. The methodaccording to claim 7, wherein the muscular disorder is a disorderresulting from a genetic mutation in a gene associated with musclefunction.
 9. The method according to claim 7, wherein the musculardisorder is Duchenne muscular dystrophy or Becker muscular dystrophy.